Compositions for enhancing DNA synthesis, DNA polymerase-related factors and utilization thereof

ABSTRACT

The invention provides methods, kits, and compositions for enhancing synthesis of DNA involving a carboxylate ion-supplying substance that is effective in promoting DNA synthesis in enzymatic DNA synthesis reactions. The invention further provides a thermostable DNA polymerase-related factor derived from  Thermococcus  species, which has an activity to promote the DNA synthesis activity of DNA polymerase or which binds to DNA polymerase.

TECHNICAL FIELD

The present invention relates to a composition for enhancing synthesisof DNA and a DNA polymerase-related factor that are useful for DNAsynthesis and DNA amplification from template nucleic acids, and so on.More specifically, the invention relates to a composition for enhancingsynthesis of DNA and a DNA polymerase-related factor that are useful forpolymerase chain reactions (PCR), and so on.

BACKGROUND ART

The DNA amplification technique represented by polymerase chainreactions (PCR); Nature, vol. 324 (6093), 1986, pp.13-19) is awell-known technique in the field of molecular biology. Detection,analysis, transcription or amplification of nucleic acids by the PCRtechnique is one of the most important operations in modern molecularbiology and is especially important in gene expression studies,diagnosis of infectious agents or hereditary diseases, cDNA production,analysis of retroviruses, etc.

Since DNA amplification performance depends on the performance of theDNA polymerase used, various DNA polymerases have been searched for innature or improved. For example, PCR was originally performed usinginsufficiently thermostable DNA polymerases derived from mesophiles suchas E. coli. However, since PCR is performed under highly stringentconditions, i.e., themocycling at temperatures in the range of about 23°C. to about 100° C. many times, the success rate (probability with whichobject DNA is successfully amplified) was low. It is now, however,common practice to utilize highly thermostable thermophile-derived DNApolymerases.

One of the important performances required of DNA polymerases is“elongation rate”. A typical method of determining DNA elongation ratecomprises performing a DNA synthesis reaction in a buffer using DNAprepared by annealing of M13 single-stranded DNA (1.6 μg) andcomplementary primer(s) (16 pmole) as a template and using KOD, Pfu,Deep Vent, Taq or like various DNA polymerases (5U) (as an enzyme),followed by calculating the DNA elongation rate from the relationshipbetween the reaction time and the size of DNA synthesized.

For example, the DNA elongation rate of KOD polymerase is 105 to 130bases/second, that of Pfu polymerase is 24.8 bases/second, that of DeepVent polymerase is 23.3 bases/second, and that of Taq polymerase is 61.0bases/second (Takagi, M. et al.: Characterization of DNA polymerase fromPyrococcus sp. KOD1 and its application to PCR; Appl. Environ.Microbiol. 63, 4504-45410, (1997)).

“Fidelity” is another important performance required of DNA polymerases.One example of a method for evaluating the DNA synthesis fidelity of DNApolymerase is using a ribosomal protein S12 (rpsL) gene associated withstreptomycin resistance. Streptomycin is an antibiotic that inhibitsprotein synthesis in prokaryote. This antibiotic binds to bacterial 30Sribosomal RNA (rRNA) to inhibit initiation complex for protein synthesisformation reactions and cause the misreading of genetic code.Streptomycin-resistant strains have a mutation at the ribosomal proteinS12 locus. This mutation is known to produce pleiotropic effects such asinhibiting suppressor tRNA from reading the stop codon to enhancetranslation fidelity of the ribosome. Thus, when PCR amplification iscarried out using rpsL gene as a template, a mutation is introduced witha certain probability. When the mutation occurs at the amino acid level,the rpsL protein structure is changed so that streptomycin cannot act on30S ribosomal RNA (rRNA). Therefore, when the amplified plasmid DNA isused to transform E. coli, the more mutations introduced, the higher thefrequency of streptomycin-resistant strain appearance.

The plasmid pMol 21 (described in Journal of Molecular Biology (1999)289, 835-850) is a plasmid containing rpsL gene and ampicillin resistantgene. A primer set for PCR amplification (one of the primers isbiotinylated and has a MluI restriction site introduced therein) isdesigned on the ampicillin resistant gene of the plasmid pMol 21 toamplify the full-length plasmid by PCR using a thermostable DNApolymerase. The obtained PCR product is purified using streptavidinbeads and cut out using the restriction enzyme MluI, followed byligating the ends using DNA ligase to transform E. coli. Thetransformant is inoculated into two types of plate media, i.e., onecontaining ampicillin, and the other containing both ampicillin andstreptomycin. The ratio of numbers of colonies appearing on the twoplate media is calculated to determine the fidelity or correctness ofDNA synthesis (Kunkel, Journal of Biological Chemistry, vol. 260, 1985,pp.5787-5796).

When PCR product fidelity of Taq DNA polymerase is determined by theabove method of determining DNA synthesis fidelity, the mutation ratewas 4% or more. In the case of a DNA polymerase capable of exhibiting3′-5′ exonuclease activity when used alone, the mutation rate was 0.05to 1%. When using a mixed enzyme of an enzyme not having 3′-5′exonuclease activity such as Taq DNA polymerase and an enzyme having3′-5′ exonuclease activity, the mutation rate was 2 to 4%. The mutationrate achieved by KOD DNA polymerase was 0.1% or less. This DNApolymerase is the most suitable enzyme for obtaining high-fidelity PCRproducts.

To what length the target DNA can be amplified (hereinafter referred toas “long-PCR performance”) is also an important requirement of DNApolymerases.

DNA polymerases, kinds of thermostable (or heat-resistant) DNApolymerases, are roughly classified into Pol I-type enzymes representedby Taq DNA polymerase and Tth DNA polymerase, and α-type enzymesrepresented by Pfu DNA polymerase. Generally, Pol I-type enzymes achievehigh DNA elongation rates but have poor fidelity because of the lack of3′-5′ exonuclease activity. In contrast, α-type enzymes, which possess3′-5′ exonuclease activity, have high fidelity but achieve low DNAelongation rates. Thus, although the two types of DNA polymerases haveexcellent properties in “elongation rate” and “fidelity”, respectively,neither have both.

With the purpose of combining the merits of both types of enzymes, Proc.Natl. Acad. Sci. USA, vol.91, 1994, pp.2216-2220 describes a techniqueutilizing a mixed type enzyme prepared by mixing two kinds ofthermostable enzymes. This enzyme mostly comprises a Pol I-type enzyme,which is considered to mainly perform DNA biosynthesis, while the α-typeenzyme merely proofreads base errors.

Japanese Patent Publication No. 3112148 describes KOD DNA polymerase, asingle enzyme (α-type enzyme) having both excellent “elongation rate”and “fidelity”.

A method using HindIII-digested λDNA labeled with ³H at the 3′ end as asubstrate and measuring the rate of ³H release under the optimaltemperatures for each polymerase is known as a typical method ofevaluating 3′-5′ exonuclease activity. More specifically, using aHindIII-digested λDNA fragment having [³H] TTP incorporated therein as asubstrate, for example, the DNA fragment and a polymerase are left in abuffer solution (20 mM Tris-HCl pH 6.5, 10 mM KCl, 6 mM (NH₄)₂SO₄, 2 mMMgCl₂, 0.1% Triton X-100, 10 μg/ml BSA) under the optimal conditions forthe polymerase and the [³H]TTP release rate is determined. The substrateDNA is prepared by adding 0.2 mM of dATP, dGTP, dCTP and [³H] TTP to 10μg of HindIII-cleaved λDNA and extending the 3′ end with Klenowpolymerase, followed by phenol extraction and ethanol precipitation torecover the DNA fragment and further purification by removal of thereleased mononucleotides using a spun column (product of Clontech).

To improve DNA amplification performance, various studies have beenconducted to improve the composition of DNA synthesis reactionsolutions. Examples of tested buffers include Tris, Tricine,bis-Tricine, HEPES, MOPS, TES, TAPS, PIPES, CAPS, etc. Examples oftested salts include potassium chloride, potassium acetate, potassiumsulfate, ammonium sulfate, ammonium chloride, ammonium acetate,magnesium chloride, magnesium acetate, magnesium sulfate, manganesechloride, manganese acetate, manganese sulfate, sodium chloride, sodiumacetate, lithium chloride, lithium acetate, etc. Examples of testedadditives include DMSO, glycerol, formamide, betaine,tetramethylammonium chloride, PEG, Tween 20, NP40, ectoine, polyols, E.coli SSB protein, phage T4 gene 32 protein and BSA, etc (PublishedJapanese Translation of PCT International Publication of PatentApplication No. H9(1997)-511133, U.S. Pat. No. 5,545,539, InternationalPublication No. WO 96/12041, U.S. Pat. No. 6,114,150, WIPO PublicationNo. WO 99/46400, Nucleic Acids Research, vol. 23, 1995, pp.3343-3344,and Nucleic Acids Research, vol. 28, 2000, p.70).

However, the above reagents are not effective for all DNA polymerases.Effectivity depends on the enzyme. Therefore, different reactionsolution compositions have been investigated for different enzymes, andoptimal reaction buffer solutions for individual enzymes have beenrecommended. It has been considered impossible to achieve a higheramplification efficiency than commercially co-packaged buffer solutions.

With the recent advances in biotechnology, the required performancelevels in DNA amplification, especially PCR, of “elongation rate”,“fidelity” and “long-PCR performance” have been raised ever upwards. Inparticular, DNA amplification fidelity has become more important in thatsuccessful elucidation of the entire human genome sequence has shiftedthe focus of study from mere detection of genes or analysis of theirdifferences to analysis of the functions of genes or proteins encoded bythe genes.

Therefore, the establishment of a DNA amplification method achievingthese requirements has been desired, but none of the prior art issatisfactory for practical use in all the requirements “elongationrate”, “fidelity” and “long-PCR performance”.

DISCLOSURE OF THE INVENTION

An object of the invention is to provide a composition for enhancingsynthesis of DNA and a DNA polymerase-related factor which enhance“elongation rate”, “fidelity” and/or “long-PCR performance” in DNAsynthesis, etc.

The present inventors have carried out intensive research to achieve theabove object and found the followings:

-   i) Anionic substances are also important in determining the success    or failure of PCR, although research in the prior art has been    carried out focusing on cations (K⁺, Na⁺, NH₄ ⁺, etc.) from the    viewpoint of their activity on nucleic acids. More specifically, PCR    success rate is enhanced by addition of carboxylate ion-supplying    substances, in particular dicarboxylic acid salts or esters, to a    PCR solution.-   ii) Among bivalent carboxylate ions, oxalate ion, malonate ion and    maleate ion are particularly effective. Among dicarboxylic acid    salts or esters, dicarboxylic acid inorganic salts are particularly    effective.-   iii) DNA amplification efficiency is further enhanced by combining    such anionic substances with at least one compound selected from the    group consisting of dimethylsulfoxide and compounds represented by    formula (1) shown below, thus achieving synergetic effects. Among    the compounds of formula (1), trimethylglycine is particularly    effective.

Successful high GC content PCR target DNA amplification and succeeds inhitherto impossible long chain target DNA amplification, etc. can bementioned as synergetic DNA amplification improvements. Moreover,enhancement in PCR amplification amount is also one of the synergisticeffects of the invention.

-   iv) The present inventors isolated a DNA polymerase-related factor    from the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1    and found that this factor can promote the DNA synthesis activity of    DNA polymerase and can bind to DNA polymerase. The inventors    succeeded in massive expression of the gene encoding the DNA    polymerase-related factor, which enables industrial scale production    of the factor.

The present invention has been accomplished based on the above findings,and provides the following compositions for enhancing synthesis of DNA,DNA polymerase-related factors, etc.

-   1. A composition for enhancing synthesis of DNA comprising at least    one anion-supplying substance that is effective in promoting DNA    synthesis in enzymatic DNA synthesis reactions.-   2. A composition for enhancing synthesis of DNA according to item 1    wherein the anion is a carboxylate ion.-   3. A composition for enhancing synthesis of DNA according to item 2    wherein the carboxylate ion-supplying substance is a salt of a    dicarboxylic acid.-   4. A composition for enhancing synthesis of DNA according to item 3    wherein the dicarboxylic acid salt is an inorganic salt.-   5. A composition for enhancing synthesis of DNA according to item 4    wherein the inorganic salt of dicarboxylic acid is an alkali metal    salt, alkaline earth metal salt or ammonium salt of dicarboxylic    acid.-   6. A composition for enhancing synthesis of DNA according to item 3    wherein the dicarboxylic acid salt is an oxalic acid salt, a malonic    acid salt or a maleic acid salt.-   7. A composition for enhancing synthesis of DNA according to item 2    wherein the carboxylate ion-supplying substance is an ester of a    dicarboxylic acid.-   8. A composition for enhancing synthesis of DNA according to item 7    wherein the dicarboxylic acid ester is an oxalic acid ester, a    malonic acid ester or a maleic acid ester.-   9. A composition for enhancing synthesis of DNA comprising:-   i) at least one anion-supplying substance that is effective in    promoting DNA synthesis in enzymatic DNA synthesis reactions; and-   ii) at least one compound selected from the group consisting of    dimethylsulfoxide and compounds represented by the following formula    R²—CH₂—NR¹ _(x)H_(y)  (1)    wherein R¹ is an alkyl group having 1 to 3 carbon atoms, R² is a    substituent selected from the group consisting of the following (a)    and (b):-   (a) ═O (oxygen) and (b) radicals represented by the formula

wherein R⁴ is methyl, hydrogen and forms a pyrrolidine ring whencombined with R¹, R⁵ is —CO₂H or —SO₃H, and n is an integer from 0 to 2,

-   -   x is an integer from 1 to 3 and    -   y is an integer from 0 to 2, provided that x plus y equals 3.

-   10. A composition for enhancing synthesis of DNA according to item 9    wherein the anion is a carboxylate ion.

-   11. A composition for enhancing synthesis of DNA according to item    10 wherein the carboxylate ion-supplying substance is a salt of a    dicarboxylic acid.

-   12. A composition for enhancing synthesis of DNA according to item    11 wherein the dicarboxylic acid salt is an inorganic salt.

-   13. A composition for enhancing synthesis of DNA according to item    12 wherein the inorganic salt is an alkali metal salt, alkaline    earth metal salt or ammonium salt.

-   14. A composition for enhancing synthesis of DNA according to item    11 wherein the dicarboxylic acid salt is an oxalic acid salt, a    malonic acid salt or a maleic acid salt.

-   15. A composition for enhancing synthesis of DNA according to item    10 wherein the carboxylate ion-supplying substance is an ester of a    dicarboxylic acid.

-   16. A composition for enhancing synthesis of DNA according to item    15 wherein the dicarboxylic acid ester is an oxalic acid ester, a    malonic acid ester or a maleic acid ester.

-   17. A composition for enhancing synthesis of DNA according to item 9    wherein the compound of formula (1) is trimethylglycine.

-   18. A composition for enhancing synthesis of DNA containing the    compound of formula (1) in an amount to achieve a concentration of    0.5 to 2M in DNA synthesis reactions, and/or containing    dimethylsulfoxide in an amount to achieve a concentration of 0.1 to    15 wt. % in DNA synthesis reactions.

-   19. A thermostable DNA polymerase-related factor derived from a    Thermococcus species, which promotes the DNA synthesis activity of a    DNA polymerase.

-   20. A DNA polymerase-related factor according to item 19 wherein the    Thermococcus species is a hyperthermophilic archaeon, Thermococcus    kodakaraensis.

-   21. A DNA polymerase-related factor according to item 20 wherein the    strain of Thermococcus kodakaraensis is Thermococcus kodakaraensis    KOD1.

-   22. A DNA polymerase-related factor according to item 19 which    promotes the DNA synthesis activity of a thermostable DNA    polymerase.

-   23. A DNA polymerase-related factor according to item 19 which    promotes the DNA synthesis activity of a DNA polymerase derived from    a Thermococcus species.

-   24. A DNA polymerase-related factor according to item 23 wherein the    Thermococcus species is the hyperthermophilic archaeon Thermococcus    kodakaraensis.

-   25. A DNA polymerase-related factor according to item 24 wherein the    strain of Thermococcus kodakaraensis is Thermococcus kodakaraensis    KOD1.

-   26. A thermostable DNA polymerase-related factor derived from a    Thermococcus species, which has an activity to bind to DNA    polymerase.

-   27. A DNA polymerase-related factor according to item 26 wherein the    Thermococcus species is the hyperthermophilic archaeon Thermococcus    kodakaraensis.

-   28. A thermostable DNA polymerase-related factor according to item    27 wherein the strain of Thermococcus kodakaraensis is Thermococcus    kodakaraensis KOD1.

-   29. A DNA polymerase-related factor according to item 26 which has    an activity to bind to a thermostable DNA polymerase.

-   30. A DNA polymerase-related factor according to item 26 which    promotes the DNA synthesis activity of a DNA polymerase derived from    a Thermococcus species.

-   31. A DNA polymerase-related factor according to item 30 wherein the    Thermococcus species is the hyperthermophilic archaeon Thermococcus    kodakaraensis.

-   32. A DNA polymerase-related factor according to item 31 wherein the    strain of Thermococcus kodakaraensis is Thermococcus kodakaraensis    KOD1.

-   33. A DNA polymerase-related factor which is KOD-PCNA (proliferating    cell nuclear antigen) derived from Thermococcus kodakaraensis KOD1,    KOD-RFCS (replication factor C small subunit) derived from    Thermococcus kodakaraensis KOD1, or KOD-RFCL (replication factor C    large subunit) derived from Thermococcus kodakaraensis KOD1.

-   34. Any one of the proteins shown in (e) to (j) below:

-   (e) a protein comprising the amino acid sequence of SEQ ID NO: 2;

-   (f) a protein which comprises an amino acid sequence resulting from    addition, deletion or substitution of one or more amino acids in the    sequence of SEQ ID NO: 2, and has an activity to promote the DNA    synthesis activity of a DNA polymerase or an activity to bind to a    DNA polymerase;

-   (g) a protein comprising the amino acid sequence of SEQ ID NO: 4;

-   (h) a protein which comprises an amino acid sequence resulting from    addition, deletion or substitution of one or more amino acids in the    sequence of SEQ ID NO: 4, and has an activity to promote the DNA    synthesis activity of a DNA polymerase or an activity to bind to a    DNA polymerase;

-   (i) a protein comprising the amino acid sequence of SEQ ID NO: 6;

-   (j) a protein which comprises an amino acid sequence resulting from    addition, deletion or substitution of one or more amino acids in the    sequence of SEQ ID NO: 6, and has an activity to promote the DNA    synthesis activity of a DNA polymerase or an activity to bind to a    DNA polymerase.

-   35. Any one of the genes shown in (k) to (p) below:

-   (k) a gene comprising the nucleotide sequence of SEQ ID NO: 3;

-   (1) a gene which hybridizes with the gene consisting of the    nucleotide sequence of SEQ ID NO: 3 under stringent conditions, and    has an activity to promote the DNA synthesis activity of a DNA    polymerase or an activity to bind to a DNA polymerase;

-   (m) a gene comprising the nucleotide sequence of SEQ ID NO: 5;

-   (n) a gene which hybridizes with the gene consisting of the    nucleotide sequence of SEQ ID NO: 5 under stringent conditions, and    has an activity to promote the DNA synthesis activity of a DNA    polymerase or an activity to bind to a DNA polymerase;

-   (o) a gene comprising the nucleotide sequence of SEQ ID NO: 7;

-   (p) a gene which hybridizes with the gene consisting of the    nucleotide sequence of SEQ ID NO: 7 under stringent conditions, and    has an activity to promote the DNA synthesis activity of a DNA    polymerase or an activity to bind to a DNA polymerase.

-   36. A process for preparing a DNA polymerase-related factor,    comprising culturing a transformant harboring the DNA of item 35 and    recovering a thermostable DNA polymerase-related factor from the    culture, the DNA polymerase-related factor having an activity to    either promote the DNA synthesis activity of a DNA polymerase or    bind to a DNA polymerase, or having both activities.

-   37. A composition for synthesizing DNA comprising the composition    for enhancing synthesis of DNA of any of items 1 to 18 and an enzyme    having DNA polymerase activity.

-   38. A composition for synthesizing DNA according to item 37 which    further comprises the DNA polymerase-related factor of any of items    19 to 33 or the protein of item 34.

-   39. A composition for synthesizing DNA according to item 37 wherein    the enzyme having DNA polymerase activity is a DNA-directed DNA    polymerase.

-   40. A composition for synthesizing DNA according to item 39 wherein    the DNA-directed DNA polymerase is selected from the group    consisting of Taq polymerase, Tth polymerase, Tli polymerase, Pfu    polymerase, Pfutubo polymerase, Pyrobest polymerase, Pwo polymerase,    KOD polymerase, Bst polymerase, Sac polymerase, Sso polymerase, Poc    polymerase, Pab polymerase, Mth polymerase, Pho polymerase, ES4    polymerase, VENT polymerase, DEEPVENT polymerase, EX-Taq polymerase,    LA-Taq polymerase, Expand polymerases, Platinum Taq polymerases,    Hi-Fi polymerase, Tbr polymerase, Tfl polymerase, Tru polymerase,    Tac polymerase, Tne polymerase, Tma polymerase, Tih polymerase, Tfi    polymerase, and variants, modified products and derivatives thereof.

-   41. A composition for synthesizing DNA according to item 39 wherein    the DNA-directed DNA polymerase is a thermostable DNA-directed DNA    polymerase which synthesizes DNA at a rate of at least 30    bases/second and has 3′-5′ exonuclease activity.

-   42. A composition for synthesizing DNA according to item 39 wherein    the DNA-directed DNA polymerase is a thermostable DNA-directed DNA    polymerase which synthesizes DNA at a rate of at least 30    bases/second and which exhibits an error rate of 4% or less when    performing PCR using mMOl 21 as a template.

-   43. A composition for synthesizing DNA according to item 37 wherein    the enzyme having DNA polymerase activity is a reverse    transcriptase.

-   44. A composition for synthesizing DNA according to item 43 wherein    the reverse transcriptase is an enzyme selected from the group    consisting of AMV-RT polymerase, M-MLV-RT polymerase, HIV-RT    polymerase, EIAV-RT polymerase, RAV2-RT polymerase, C.    hydrogenoformans DNA polymerase, SuperScript I, SuperScript II, and    variants, modified products and derivatives thereof.

-   45. A composition for synthesizing DNA according to item 43 wherein    the reverse transcriptase is an enzyme with substantially reduced    RnaseH activity.

-   46. A composition for synthesizing DNA according to item 37, further    comprising at least one member selected from the group consisting of    nucleotides, nucleotide derivatives, buffers, salts, template    nucleic acids and primers.

-   47. A composition for synthesizing DNA according to item 46 wherein    the nucleotides are deoxyphosphonucleotides and nucleotide    derivatives are deoxyphosphonucleotide derivatives.

-   48. A composition for synthesizing DNA according to item 47 wherein    the deoxyphosphonucleotides and derivatives thereof are selected    from the group consisting of dATP, dCTP, dGTP, dTTP, dITP, dUTP,    α-thio-dNTPs, biotin-dUTP, fluorescein-dUTP and digoxigenin-dUTP.

-   49. A method for synthesizing DNA comprising the steps of:

-   (a) mixing a template nucleic acid with the composition of any of    claims 8 to 15, a nucleotide and/or a nucleotide derivative, and    primers to form a mixture; and

-   (b) incubating the mixture under such conditions that DNA is    synthesized at a rate of at least 30 bases/second and, when    performing PCR using pMol 21 as a template, the error rate is 4% or    less to prepare a first nucleic acid molecule complementary to the    entire or part of the template nucleic acid.

-   50. A method for synthesizing DNA according to item 49 wherein the    template nucleic acid is a purified nucleic acid.

-   51. A method for synthesizing DNA according to item 50 wherein the    template nucleic acid is a nucleic acid purified by a method    comprising the following steps:

-   (i) mixing a nucleic acid-binding magnetic carrier consisting of    ferromagnetic metal oxide-containing magnetic silica particles, a    nucleic acid-containing material and a nucleic acid extraction    solution;

-   (ii) separating the nucleic acid-bound magnetic carrier from the    residual mixture using a magnetic field; and

-   (iii) eluting the nucleic acid from the magnetic carrier.

-   52. A method for synthesizing DNA according to item 49 further    comprising the step of (c) incubating a mixture containing the first    nucleic acid molecule under such conditions that DNA is synthesized    at a rate of at least 30 bases/second and, when performing PCR using    pMol 21 as a template, the error rate is 4% or less to prepare a    second nucleic acid molecule complementary to the entire or part of    the first nucleic acid molecule.

-   53. A method for synthesizing DNA according to item 49 further    comprising the step of (d) purifying the synthesized DNA.

-   54. A method for synthesizing DNA according to item 53 wherein the    DNA purification step (d) comprises the following steps:

-   (iv) mixing a nucleic acid-binding magnetic carrier consisting of    ferromagnetic metal oxide-containing magnetic silica particles, a    material comprising the synthesized DNA, and a nucleic acid    extraction solution;

-   (v) separating the DNA-bound magnetic carrier from the residual    mixture using a magnetic field; and

-   (vi) eluting the nucleic acid from the magnetic carrier.

-   55. A method for synthesizing DNA according to any of items 49 to 54    using hot start PCR.

-   56. A nucleic acid molecule obtained by the method of item 49.

-   57. A DNA amplification method comprising the steps of:

-   (a) mixing a template nucleic acid with the composition of any of    items 37 to 45, nucleotide and/or nucleotide derivatives and primers    to form a mixture; and

-   (b) incubating the mixture under such conditions that DNA is    synthesized at a rate of at least 30 bases/second and, when    performing PCR using pMol 21 as a template, the error rate is 4% or    less to amplify a nucleic acid molecule complementary to the entire    or part of the template nucleic acid.

-   58. A DNA amplification method according to item 57 wherein the    template nucleic acid is a purified nucleic acid.

-   59. A DNA amplification method according to item 58 wherein the    template nucleic acid is a nucleic acid purified by a method    comprising the following steps:

-   (i) mixing a nucleic acid-binding magnetic carrier consisting of    magnetic silica particles containing ferromagnetic metal oxide, a    nucleic acid-containing material, and a nucleic acid extraction    solution;

-   (ii) separating the nucleic acid-bound magnetic carrier from the    residual mixture using a magnetic field; and

-   (iii) eluting the nucleic acid from the magnetic carrier.

-   60. A DNA amplification method according to item 57 further    comprising the step of (c) incubating a mixture containing the first    nucleic acid molecule under such conditions that DNA is synthesized    at a rate of at least 30 bases/second and, when performing PCR using    pMol 21 as a template, the error rate is 4% or less to prepare a    second nucleic acid molecule complementary to the entire or part of    the first nucleic acid.

-   61. A DNA amplification method according to item 57 further    comprising the step of (d) purifying the synthesized DNA.

-   62. A DNA amplification method according to item 61 wherein the DNA    purification step (d) comprises the following steps:

-   (iv) mixing a nucleic acid-binding magnetic carrier consisting of    ferromagnetic metal oxide-containing magnetic silica particles, a    material comprising the synthesized DNA, and a nucleic acid    extraction solution;

-   (v) separating the DNA-bound magnetic carrier from the residual    mixture using a magnetic field; and

-   (vi) eluting the nucleic acid from the magnetic carrier.

-   63. A DNA amplification method according to any of items 57 to 62    using hot start PCR.

-   64. A method for nucleotide sequencing comprising the steps of:    -   (a) mixing a target nucleic acid with the composition of any of        items 37 to 45, nucleotide and/or nucleotide derivatives,        primers and a release factor to form a mixture;    -   (b) incubating the mixture under such conditions that DNA is        synthesized at a rate of at least 30 bases/second and, when        performing PCR using pMol 21 as a template, the error rate is 4%        or less to amplify a nucleic acid molecule complementary to the        entire or part of the target nucleic acid; and    -   (e) separating the amplified nucleic acid molecule to determine        the entire or partial nucleotide sequence.

-   65. A method for nucleotide sequencing according to item 64 wherein    the target nucleic acid is a purified nucleic acid.

-   66. A method for nucleotide sequencing according to item 65 wherein    the target nucleic acid is a nucleic acid purified by a method    comprising the following steps:

-   (i) mixing a nucleic acid-binding magnetic carrier consisting of    ferromagnetic metal oxide-containing magnetic silica particles, a    nucleic acid-containing material and a nucleic acid extraction    solution;

-   (ii) separating the nucleic acid-bound magnetic carrier from the    residual mixture using a magnetic field; and

-   (iii) eluting the nucleic acid from the magnetic carrier.

-   67. A method for nucleotide sequencing according to item 64 further    comprising, between steps (b) and (e) (or between steps (b) and (d)    when the process further comprises step (d)), the step of (c)    incubating a mixture containing a first nucleic acid molecule under    such conditions that DNA is synthesized at a rate of at least 30    bases/second and, when performing PCR using pMol 21 as a template,    the error rate is 4% or less to prepare a second nucleic acid    molecule complementary to the entire or part of the first nucleic    acid molecule.

-   68. A method for nucleotide sequencing according to item 64 further    comprising, between steps (b) and (e) (or between steps (c) and (e)    when the process further comprises step (c)), the step of (d)    purifying the synthesized DNA.

-   69. A method for nucleotide sequencing according to item 68 wherein    the DNA purification step (d) comprises the following steps:

-   (iv) mixing a nucleic acid-binding magnetic carrier consisting of    ferromagnetic metal oxide-containing magnetic silica particles, a    material comprising the synthesized DNA, and a nucleic acid    extraction solution;

-   (v) separating the DNA-bound magnetic carrier from the residual    mixture using a magnetic field; and

-   (vi) eluting the nucleic acid from the magnetic carrier.

-   70. A method for nucleotide sequencing according to any of items 64    to 69 using hot start PCR.

-   71. A kit for synthesizing DNA molecule comprising the DNA    synthesizing composition of any of items 37 to 48.

-   72. A kit for synthesizing DNA molecule according to item 71 wherein    the components are entirely contained in one part or separately    contained in two or more parts.

-   73. A method for synthesizing DNA comprising reacting a DNA    polymerase with a template nucleic acid in the presence of the DNA    polymerase-related factor of any of items 19 to 33 or the protein of    item 34.

-   74. A method for synthesizing DNA according to item 73 using at    least two members selected from the group consisting of the DNA    polymerase-related factors of items 19 to 33 and proteins of item    34.

-   75. A DNA synthesis method according to item 73 wherein the DNA    polymerase is a thermostable DNA polymerase.

-   76. A DNA synthesis method according to item 73 which is a PCR    method.

-   77. A kit for synthesizing DNA comprising a DNA polymerase and the    DNA polymerase-related factor of any of items 19 to 33 or the    protein of item 34.

-   78. A kit for synthesizing DNA according to item 77 wherein the DNA    polymerase is a thermostable DNA polymerase.

According to the invention, hitherto impossible target nucleic acid DNAsyntheses become possible by utilizing an anion-supplying substanceeffective in promoting DNA synthesis. The invention enables not onlysimple DNA synthesis but also PCR to be performed on a target nucleicacid whose PCR was conventionally impossible, thus enhancing the PCRsuccess rate. These effects are obtainable with all kinds of DNApolymerases.

According to the invention, the success rate of DNA synthesis,particularly the PCR success rate, is further enhanced by using thecompound of formula (1) and/or DMSO in combination with the anioneffective in promoting DNA synthesis, thus achieving synergetic effects.These synergetic effects are confirmed with several DNA polymerases andare effective for not only simple DNA synthesis reactions but also PCRmethods. In particular, long PCR becomes possible even with α-type DNApolymerases which are considered to be unsuitable for long PCR. Further,by using hot start PCR, stable effectiveness for long PCR can beachieved.

Moreover, the invention enables mass production of DNApolymerase-related factor that enhances DNA synthesis activity of DNApolymerases. The DNA synthesis activity-promoting effect of the DNApolymerase-related factor is effective for not only simple DNA synthesisreactions but also PCR methods, thus making conventionally impossiblePCR possible beyond the prior limitations, with each DNA polymerase, onthe probability of PCR success, amplification amount enhancement andfidelity enhancement.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is described below in detail.

(I) First Enhancing Composition for DNA Synthesis

The first enhancing composition for DNA synthesis of the invention is acomposition for enhancing synthesis of DNA comprising at least oneanion-supplying substance that is effective in promoting DNA synthesisin enzymatic DNA synthesis reactions.

Anion-Supplying Substance

The anion-supplying substance (anionic substance) that is effective inpromoting DNA synthesis is not particularly limited and includessubstances that have carboxyl groups and supply carboxylate ions. Saltsand esters of dicarboxylic acids are preferable, and salts ofdicarboxylic acids are particularly preferable. Inorganic salts ofdicarboxylic acid salts are preferable, and alkali metal salts, alkalineearth metal salts and ammonium salts are particularly preferable. Amongthese, alkali metal salts are especially preferable and potassium saltsor sodium salts are particularly preferable.

Usable dicarboxylic acids are not particularly limited and preferableexamples include oxalic acid, malonic acid and maleic acid. Oxalic acidis particularly preferable.

Examples of dicarboxylic acid salts or esters include zinc oxalate,ammonium oxalate, potassium oxalate, calcium oxalate, diethyl oxalate,N,N′-disuccinimidyl oxalate, dimethyl oxalate, tin oxalate, ceriumoxalate, iron oxalate, copper oxalate, sodium oxalate, nickel oxalate,bis oxalate, 2,4-dinitrophenyl oxalate, 2,4,6-trichlorophenyl oxalate,manganese oxalate, methyl oxalate, lanthanum oxalate, lithium oxalate,isopropylidene malonate, ethyl malonate, diethyl malonate, dibenzylmalonate, dimethyl malonate, thallium malonate, disodium malonate,monosodium maleate, diethyl maleate, chlorpheniramine maleate,di-n-butyl maleate, mono-n-butyl maleate; and the like. All thesecompounds are commercially available.

Preferably, the composition for enhancing synthesis of DNA contains suchan anion-supplying substance in an amount to achieve a concentration inthe DNA synthesis reaction solution of usually about 0.1 to 20 mM,preferably about 0.1 to 10 mM, and more preferably about 1 to 10 mM.

(II) Second Enhancing Composition for DNA Synthesis

The second enhancing composition for DNA synthesis of the invention is acomposition for enhancing synthesis of DNA comprising:

-   i) at least one anion-supplying substance that is effective in    promoting DNA synthesis in enzymatic DNA synthesis reactions; and-   ii) at least one compound selected from the group consisting of    dimethylsulfoxide and compounds represented by the following formula    (1):    R²—CH₂—NR¹ _(x)H_(y)  (1)    wherein R¹ is an alkyl group having 1 to 3 carbon atoms, R² is a    substituent selected from the group consisting of the following (a)    and (b):-   (a) oxygen (═O) and (b) radicals represented by the formula

wherein R⁴ is methyl or hydrogen, and forms a pyrrolidine ring whencombined with R¹, R⁵ is —CO₂H or —SO₃H, and n is an integer from 0 to 2,x is an integer from 1 to 3 and y is an integer from 0 to 2, providedthat x plus y equals 3.Compound of Formula (1)

In the compound of formula (1), R¹ includes methyl, ethyl, n-propyl,isopropyl and the like. Methyl is particularly preferable. R² ispreferably carboxyl. Especially preferable are trialkylglycines whereinx is 3 and y is 0. Particularly preferable is trimethylglycine(betaine). Commercially available trimethylglycines can be used.

The effective concentration of the compound of formula (1) in the DNAsynthesis reaction solution is usually about 0.5 to 2M. Therefore, thecomposition for enhancing synthesis of DNA preferably contains thecompound in an amount to achieve a concentration in the DNA synthesisreaction solution of about 0.5 to 2M, preferably about 0.5 to 1.5M, andmore preferably 1 to 1.5M.

DMSO

Commercially available DMSO, etc. can be used. The effectiveconcentration of DMSO in the DNA synthesis reaction system is usuallyabout 0.1 to 15 volume %. Therefore, the composition for enhancingsynthesis of DNA preferably contains DMSO in an amount to achieve aconcentration in the DNA synthesis reaction of about 0.1 to 15 wt. %,preferably about 2 to 10 wt. %, and more preferably about 5 to 10 wt. %.

Combination

The second enhancing composition for DNA synthesis of the inventioncomprises the compound of formula (1) and/or DMSO, in addition to theanionic substance, thus achieving further enhanced DNA synthesispromoting effects. The second composition for enhancing synthesis of DNAis not specifically limited but preferably comprises the above threecomponents, i.e., the anionic substance, the compound of formula (1) andDMSO.

Preferable combinations of the components of the second enhancingcomposition for DNA synthesis are, for example, a combination of anoxalic acid salt or ester (particularly a salt) and trimethylglycine, acombination of a malonic acid salt or ester (particularly a salt) andtrimethylglycine, a combination of a maleic acid salt or ester(particularly a salt) and trimethylglycine, a combination of an oxalicacid salt or ester (particularly a salt) and DMSO, a combination of amalonic acid salt or ester (particularly a salt) and DMSO, a combinationof a maleic acid salt or ester (particularly a salt) and DMSO, acombination of an oxalic acid salt or ester (particularly a salt),trimethylglycine and DMSO, a combination of a malonic acid salt or ester(particularly a salt), trimethylglycine and DMSO, a combination of amaleic acid salt or ester (particularly a salt), trimethylglycine andDMSO, and the like. The combination of an oxalic acid salt or ester(particularly a salt) and trimethylglycine is particularly preferable.

For determining the combination of types of components and most suitableconcentrations, the type of target DNA, size of the DNA fragment to beamplified, composition of DNA synthesis reaction buffer solution, andthe like may need to be considered.

(III) Composition for DNA Synthesis

The DNA synthesizing composition of the invention is a compositioncomprising an enzyme having DNA polymerase activity and the firstcomposition for enhancing synthesis of DNA or second composition forenhancing synthesis of DNA of the invention described above.

The enzyme having DNA polymerase activity (DNA polymerase) is notparticularly limited and examples include DNA-directed DNA polymerasesand reverse transcriptases. The DNA polymerase is preferablythermostable. Commercially available enzymes can be used. The reactionsolution composition of the invention may contain a plurality of enzymetypes having different DNA polymerase activities.

DNA-Directed DNA Polymerase

DNA-directed DNA polymerases are roughly classified into Pol I-type DNApolymerases, α-type DNA polymerases and mixed-type polymerases. It isgenerally thought that Pol I-type enzymes achieve high DNA elongationrates but have poor fidelity because of the lack of 3′-5′ exonucleaseactivity, whereas α-type enzymes have high fidelity owing to their 3′-5′exonuclease activity but achieve low DNA elongation rates. It ispossible to form mixed-type enzymes by mixing a α-type DNA polymeraseand a Pol I-type DNA polymerase.

Representative examples of Pol I-type enzymes are Taq DNA polymerase andTth DNA polymerase. Pol I-type enzymes also include Platinum Taq DNApolymerase series. Examples of α-type enzymes include Tli polymerase,Pfu polymerase, Pfutubo polymerase, Pyrobest polymerase, Pwo polymerase,KOD polymerase, Bst polymerase, Sac polymerase, Sso polymerase, Pocpolymerase, Pab polymerase, Mth polymerase, Pho polymerase, ES4polymerase, VENT polymerase, DEEPVENT polymerase and the like. Themixed-type enzymes include EX-Taq polymerase, LA-Taq polymerase, Expandpolymerase series, Hi-Fi polymerase and the like. Unclassified types ofenzymes include, for example, Tbr polymerase, Tfl polymerase, Trupolymerase, Tac polymerase, Tne polymerase, Tma polymerase, Tihpolymerase, Tfi polymerase and the like. Variants, modified products andderivatives thereof are also usable.

Of these enzymes, Taq, Platinum Taq, Tth, Tli, Pfu, Pfutubo, Pyrobest,Pwo and KOD, VENT, DEEPVENT, EX-Taq, LA-Taq, the Expand series andPlatinum Taq Hi-Fi are all commercially available. The other enzymes canbe readily isolated from specific bacteria by those of ordinary skill inthe art.

The DNA-directed DNA polymerase contained in the DNA synthesizingcomposition of the invention is preferably a thermostable DNA-directedDNA polymerase which synthesizes DNA at a rate of at least 30bases/second and has 3′-5′ exonuclease activity. The DNA elongation ratecan be measured, for example, by the above-mentioned method.

Also preferably, the polymerase is a thermostable DNA-directed DNApolymerase which synthesizes DNA at a rate of at least 30 bases/secondand which exhibits an error rate of 4% or less when performing PCR usingpMol 21 as a template.

Enzymes that exhibit an error rate of not higher than 1% in PCR usingpMol 21 as a template are especially preferable as DNA-directed DNApolymerases. Enzymes that are capable of amplifying at least 20 kb ofthe template DNA fragment are especially preferable. The method ofdetermining the error rate in PCR using pMol 21 as a template is asmentioned above.

The DNA-directed DNA polymerase can be suitably selected in view of theproperties such as “elongation rate”, “fidelity”, “long-PCR performance”and “heat resistance”. A combination of enzymes can also be used.

Since DNA synthesis fidelity depends mainly on the presence or absenceof 3′-5′ exonuclease activity, enzymes with 3′-5′ exonuclease activity(e.g., α-type enzymes) are preferable from the viewpoint of fidelity.

Among them, Thermococcus kodakaraensis KOD1-derived KOD polymerase isparticularly preferable because it also has excellent properties otherthan just fidelity. KOD polymerase is a thermostable DNA polymerase thatsynthesizes DNA at a rate of at least 30 bases/second, has 3′-5′exonuclease activity and exhibits an error rate of not higher than 1%when performing PCR using pMol as a template. KOD polymerase is alsocapable of amplifying 20 kb or more of target DNA. As KOD polymeraseamino acid sequence, the amino acid sequence of SEQ ID NO: 1 is known.KOD polymerases which may be contained in the DNA synthesizingcomposition of the invention include not only protein consisting of theamino acid sequence of SEQ ID NO: 1 but also modified products thereofsubstantially maintaining the above properties. More specifically, theDNA polymerases include modified proteins that consist of an amino acidsequence resulting from addition, deletion or substitution of one ormore amino acids in the sequence of SEQ ID NO: 1, and substantiallypossess the above-mentioned properties.

Reverse Transcriptase

The enzyme having DNA polymerase activity may be a reversetranscriptase. Examples of reverse transcriptases include AMV-RTpolymerase, M-MLV-RT polymerase, HIV-RT polymerase, EIAV-RT polymerase,RAV2-RT polymerase, C. hydrogenoformans DNA polymerase, SuperScript I,SuperScript II, and variants, modified products and derivatives thereof.The RNaseH activity of reverse transcriptase may be substantiallysuppressed by modification, etc. Enzymes with substantially suppressedRnase activity include, for example, those in which the Rnase activityis reduced to 90% or less, and preferably those in which the Rnaseactivity is reduced to 10% or less.

Other Components

The DNA synthesizing composition of the invention may further contain,in addition to the DNA synthesis promotor and enzyme(s) having DNApolymerase activity, at least one member selected from the groupconsisting of template nucleic acids, one or more types ofoligonucleotide primers, one or more types of nucleotides, nucleotidederivatives, buffers, salts, and additives useful for DNA synthesis.

The template nucleic acid may be DNA, RNA, mixtures thereof, etc. Usableoligonucleotide primers include forward primers and backward primershaving a nucleotide sequence complementary to a part of the targetnucleic acid.

The nucleotides or derivatives thereof are not particularly limited. Forexample, deoxyphosphonucleotides or derivatives thereof can be used.Specific examples include dATP, dCTP, dGTP, dTTP, dITP, dUTP,α-thio-dNTPs, biotin-dUTP, fluorescein-dUTP and digoxigenin-dUTP.

The salts contained in the reaction solution composition of theinvention are not particularly limited. Examples thereof includesubstances selected from the group consisting of potassium chloride,potassium acetate, potassium sulfate, ammonium sulfate, ammoniumchloride, ammonium acetate, magnesium chloride, magnesium acetate,magnesium sulfate, manganese chloride, manganese acetate, manganesesulfate, sodium chloride, sodium acetate, lithium chloride and lithiumacetate.

Usually the DNA synthesizing composition contains salts capable ofsupplying magnesium ions such as magnesium chloride, magnesium acetateor magnesium sulfate. These salts are commercially available.Preferably, magnesium ions are contained in the DNA synthesizingcomposition in such a concentration that their concentration in the DNAsynthesis reaction solution becomes about 0.5 to 10 mM, and morepreferably about 1 to 3 mM.

Salts can enhance the solubility of DNA or proteins by increasing theionic strength of the reaction solution. In addition, salts can alsoenhance the annealing efficiency of oligonucleotides to nucleic acids.

Buffers are not particularly limited. Examples of usable buffers includeTRIS, TRICINE, bis-Tricine, HEPES, MOPS, TES, TAPS, PIPES and CAPS.

Preferably, the buffer is contained in the DNA synthesizing compositionin such an amount that its concentration in the DNA synthesis reactionsolution becomes about 10 to 200 mM, and more preferably about 20 to 100mM.

Examples of additives useful for DNA synthesis include formamide,tetramethylammonium chloride, glycerol, PEG, Tween-20, NP40, ectoine,polyols, Escherichia coli SSB protein, phage T4 gene 32 protein, BSA andthe like. Tetramethylammonium chloride is considered to promote thespecificity of primers. BSA is considered to improve the stability ofenzymes.

One example of a DNA synthesizing composition of the invention is acomposition comprising 20 mM Tris-HCl (pH 6.5 at 75° C.), 10 mM KCl, 6mM (NH₄)₂SO₄, 1 to 3 mM MgCl₂, 0.1% Triton X-100, 10 μg/ml BSA, 20 to200 μM dNTPs, 0.1 pM to 1 μM primer, and 0.1 to 250 ng template DNA.

The DNA synthesizing composition of the invention may contain labelednucleic acid probes, color reagents, luminescence reagents and the like.These additives may be added after DNA amplification. Such nucleic acidprobes, color reagents, luminescence reagents, etc. will be describedlater.

The DNA synthesizing composition of the invention may further containantibodies against the DNA polymerase, antibodies for hot start, knownenhancers and the like. Antibodies against DNA polymerases are used tosuppress the production of byproducts such as primer dimers bycompletely suppressing the enzyme activity at room temperature.Antibodies for hot start are used for methods (i.e., hot start PCRmethods) enhancing the PCR success rate by protecting the primer fromdigestion.

DNA Polymerase-Related Factor

The DNA synthesizing composition of the invention may contain DNApolymerase-related factors as described later.

(IV) First DNA Synthesis Method

The first DNA synthesis method of the invention comprises the steps of:

-   (a) mixing a template nucleic acid, the above DNA synthesizing    composition of the invention (DNA synthesizing composition    comprising the composition for enhancing synthesis of DNA of the    invention and an enzyme having DNA polymerase activity), nucleotides    or nucleotide derivatives and primers to form a mixture; and-   (b) incubating the mixture under such conditions that DNA is    synthesized at a rate of at least 30 bases/second and, when    performing PCR using pMol 21 as a template, the error rate is 4% or    less to prepare a first nucleic acid molecule complementary to the    entire or part of the template nucleic acid.

The present invention includes nucleic acid molecules prepared by theabove method.

In the first DNA synthesis method of the invention, when DNA is used asa template nucleic acid as in standard PCR, the target DNA molecule isobtained as the first nucleic acid molecule. When RNA is used as atemplate nucleic acid as in RT-PCR, cDNA is obtained as the firstnucleic acid molecule. When RNA is used as a template nucleic acid, thetarget DNA molecule can be synthesized as a second nucleic acid moleculeafter obtaining cDNA as the first nucleic acid molecule, by the methoddescribed later.

In step (b) of the above method, it is preferable to use conditions suchthat the error rate during PCR using pMol 21 as a template is 1% orless. In addition, it is preferable to use conditions such that target20 kb or more DNA in template nucleic acid can be amplified.

In step (a), the mixture may further contain buffers, salts and otheradditives useful for DNA synthesis.

In the first DNA synthesis method of the invention, purified orunpurified template nucleic acids may be used but a purified one ispreferable. In particular, when the supply source of nucleic acid iscells from tissues; cells from body fluids such as blood, lymph, milk,urine, sperm and the like; feces; cultured cells; cells in agarose orpolyacrylamide, it is preferable to purify the nucleic acid becausecontaminants that interfere with the treatment or analysis in thenucleic acid synthesis or amplification step and/or subsequent steps arelikely to be present in substantial amounts.

Examples of such contaminants include substances that interfere with orsuppress chemical reactions (such as nucleic acid or proteinhybridization, enzymatic catalytic reactions, etc.); substances thatcatalyze the degradation, digestion or polymerization of the targetnucleic acid or other biological substances; and substances that give afalse positive background in target substance-quantification samples incases where no nucleic acid is actually present in the sample.

Specific examples of contaminants include high polymers, or highmolecular materials from in vivo or in vitro media from which the targetnucleic acid is isolated, enzymes and like high molecular weightmaterials, other types of proteins, polysaccharides, polynucleotides,lipids and like low molecular weight materials, low molecular weightenzyme inhibitors, oligonucleotides and the like. Contaminants may beintroduced into the target biological substance from chemicals orsubstances used to isolate the target nucleic acid. Trace of metals,dyes and organic solvents are examples of such contaminants.

From another viewpoint, when 10 kb or more, and preferably 20 kb or moreof the target DNA is intended to be synthesized by long PCR, it ispreferable to purify the template nucleic acid to reduce the possibilityof reaction stoppage due to the effects of contaminants. Because RNAneeds to be isolated in RT-PCR, a purified template nucleic acid ispreferably used. In standard PCR or similar reactions other than thosementioned above, it is also preferable to purify the template nucleicacid to prevent the presence of substantial levels of contaminants thatinterfere with polymerase reactions.

The purification method is not particularly limited. However, thetemplate nucleic acid is preferably purified by a method comprising thefollowing steps (i) to (iii) because of its convenience, high safety,economy, high yields, relatively mild treatment conditions, etc., ascompared with known nucleic acid purification methods such asphenol/chloroform extraction/ethanol precipitation methods.

-   -   Step (i): mixing a nucleic acid-binding magnetic carrier        consisting of ferromagnetic metal oxide-containing magnetic        silica particles, a nucleic acid-containing material and a        nucleic acid extraction solution;    -   Step (ii): separating the nucleic acid-bound magnetic carrier        from the residual mixture using a magnetic field; and    -   Step (iii): eluting the nucleic acid from the magnetic carrier.

The DNA synthesis method of the invention may further contain the step(c) of incubating a mixture containing the first nucleic acid moleculeunder such conditions that DNA is synthesized at a rate of at least 30bases/second, and when performing PCR using pMol 21 as a template, theerror rate is 4% or less to prepare a second nucleic acid moleculecomplementary to the entire or part of the first nucleic acid molecule.

When DNA is used as the template nucleic acid, step (c) is a process ofsynthesizing DNA as a second nucleic acid molecule by using the DNAobtained as a first nucleic acid molecule in steps (a) and (b) as atemplate. When RNA is used as the template nucleic acid, step (c) is aprocess of synthesizing DNA as a second nucleic acid molecule by usingcDNA obtained as a first nucleic acid molecule in steps (a) and (b) as atemplate.

In step (c), it is also preferable to use conditions such that the errorrate during PCR using pMol 21 as a template is 1% or less. In addition,it is preferable to use conditions such that 20 kb or more of firstnucleic acid target DNA can be amplified.

The DNA synthesis method of the invention may further contain the step(d) of purifying the obtained first nucleic acid molecule. Thepurification step (d) may comprise, for example, the following steps(iv) to (vi):

-   -   (iv) mixing a nucleic acid-binding magnetic carrier consisting        of ferromagnetic metal oxide-containing magnetic silica        particles, a material comprising the synthesized DNA, and a        nucleic acid extraction solution;    -   (v) separating the DNA-bound magnetic carrier from the residual        liquid using a magnetic field; and    -   (vi) eluting the nucleic acid from the magnetic carrier.

Since the above purification method is carried out using mild treatmentconditions, it is rare that nucleic acid is mechanically sheared, thusbeing preferable, particularly when the template nucleic acid issubjected to long-PCR. After performing DNA synthesis, it is sometimesnecessary to isolate the target substance from the other components inthe solution. In this case also, the use of nucleic acid isolation orpurification methods similar to the above is effective.

In the first DNA synthesis method of the invention, hot start PCRmethods can be used.

Hot start PCR is used to avoid the occurrence of problems such asnon-specific formation of double-stranded hybride from a primer and atemplate DNA, formation of double-stranded hybrids from primers to causean extra band or primer dimer to form, and reduction of the specificityof primers by digestion of the primers with enzymes having 3′-5′exonuclease activity when such enzymes are used; and these problemsoccurring upon raising the temperature in the initial denaturationheating step to convert double stranded DNA into a single-stranded DNAprior to the temperature cycling in the PCR process.

The hot start PCR method is not particularly limited. Methods comprisingadding an enzyme after the temperature rise and methods using wax toseparate the enzyme from the reaction solution are convenient. However,the former has cross-contamination and operative problems due to theopening and closing of the tubes, whereas the latter has a problem inthat immediately after melting of the wax, the enzyme does not uniformlyexist in the reaction medium. Another method comprises hydrolysing theenzyme using a reagent but it takes a long time, i.e., about 10 to 20minutes to inactivate the reagent.

Currently, a method comprising blocking the enzymatic activity using anantibody against DNA polymerase is preferably used a convenient andreproducible method. According to this method, the antibody binds to theenzyme in a preprepared reaction solution to inactivate the enzyme, andwhen the temperature exceeds a certain level during heating in theinitial denaturation step, the antibody is denatured so that the enzymeis activated and PCR starts.

In the case of using RNA as a template nucleic acid, i.e., RT-PCR, inorder to allow hot start PCR to proceed in the step of synthesizing DNAas a second nucleic acid molecule using cDNA obtained as a first nucleicacid in steps (a) and (b) as a template, whether the antibody is addedat the beginning of the reverse-transcription reaction or after cDNAsynthesis can be suitably decided in consideration of thereverse-transcription reaction temperature and whether all the steps aresequentially performed as a single continuous step or divided into twosteps, i.e., a cDNA synthesis step and the subsequent steps.

(V) DNA Amplification Method

The DNA amplification method of the invention comprises the steps of:

-   (a) mixing a template nucleic acid with the DNA synthesizing    composition of the invention (DNA synthesizing composition    comprising the composition for enhancing synthesis of DNA of the    invention and an enzyme having DNA polymerase activity), nucleotides    or nucleotide derivatives and primers to form a mixture; and-   (b) incubating the mixture under such conditions that DNA is    synthesized at a rate of at least 30 bases/second and, when    performing PCR using pMol 21 as a template, the error rate is 4% or    less, to amplify a nucleic acid molecule complementary to the entire    or part of the template nucleic acid.

In step (b), it is preferable to use conditions such that the error ratein PCR using pMol 21 as a template is 1% or less. In addition, it ispreferable to use conditions such that target 20 kb or more DNA intemplate nucleic acid can be amplified.

In the above DNA synthesis method, a purified nucleic acid is preferablyused as a template. The purification method of the template nucleic acidis as described above in the DNA synthesis method of the invention.

The DNA amplification method of the invention may further comprise thestep of (c) incubating a mixture containing the first nucleic acidmolecule under such conditions that DNA is synthesized at a rate of atleast 30 bases/second, and when performing PCR using pMol 21 as atemplate the error rate is 4% or less to prepare a second nucleic acidmolecule complementary to the entire or part of the first nucleic acid.

The DNA amplification method of the invention may further comprise thestep of (d) purifying the amplified nucleic acid molecule. Thepurification step is also as described above in the DNA synthesis methodof the invention.

Hot start PCR can also be used in the DNA amplification method of theinvention.

PCR

Examples of the enzymatic reaction for DNA synthesis used in the DNAamplification method of the invention include DNA-directed DNApolymerase reactions and reverse transcriptase reactions. Among these,DNA-directed DNA polymerase reactions, in particular gene amplificationmethods using PCR are especially effective.

Gene amplification methods using PCR are methods which compriserepeating a 3-step cycle consisting of denaturation, annealing andelongation in the presence of a template nucleic acid, the 4 types ofdeoxyribonucleoside triphosphates, a pair of primers and a DNApolymerase to exponentially amplify the target DNA region flanked by thepair of primers (Nature, 324 (6093), 13-19 (1986)). More specifically, anucleic acid sample is denatured in the denaturation step; in thesubsequent annealing step, each primer is hybridized to itscomplementary region on the single-stranded template nucleic acid; andin the subsequent elongation step, new DNA chains complementary to thesingle-stranded target nucleic acid region are elongated from eachprimer by the action of DNA polymerase to form double-stranded DNA. Onedouble-stranded DNA fragment is amplified to two double-stranded DNAfragments per cycle. Therefore, if this cycle is repeated n times, thesample DNA region between the pair of primers is theoretically amplified2^(n) times.

The reaction conditions preferable for amplification are such thatthermocycling conditions, i.e., the temperature of the reaction mixtureis changed to perform each step of the PCR cycle. Thermocycling isusually performed at a temperature in the range of about 23° C. to about100° C., and preferably about 37° C. to about 95° C. Nucleic aciddenaturation is usually performed at a temperature in the range fromabout 90° C. to about 100° C., and preferably about 94° C. Annealing isusually performed at a temperature in the range from about 37° C. toabout 75° C., and preferably about 60° C. DNA elongation is usuallyperformed at a temperature in the range from about 55° C. to about 80°C., and preferably about 68° C. to about 72° C.

The number of cycles varies greatly depending on desired amount of theDNA product. The number of PCR cycles is preferably in the range fromabout 5 to about 99 cycles, especially preferably about 20 cycles ormore, and particularly preferably about 25 to about 40 cycles.

Detection of Amplified Product

In the invention, the target nucleic acid can be detected by, forexample, using a labeled probe to detect the amplified product obtainedby the above amplification reaction. The labeled probe is anoligonucleotide which has a nucleotide sequence complementary to thelabeled nucleic acid and to which is attached a marker or marker-boundsubstance.

Examples of usable markers include alkaline phosphatase, peroxidase,galactosidase and like enzymes, fluorescent or radioactive substancesand the like. Examples of usable marker-binding substances includebiotin, digoxigenin and the like. Markers may be bound via biotin,digoxigenin, avidin, etc. One example of a method of introducing thesemarkers into probes is synthesis of oligonucleotide using, as onecomponent of dNTP, dNTP to which are attached such markers ormarker-bound substances. When enzymes are used as marker substances,color reagents, i.e., substrates that exhibit color upon digestion withenzymes (e.g., tetramethylbenzidine (TMB) or ortho-phenylenediamine(OPD)), or luminescent reagents, i.e., substrates emitting light (e.g.,CDP-Star, PPD)) are usable.

The detection of labeled probe-bound nucleic acids can be performed byknown methods, such as Southern hybridization or Northern hybridizationmethods. These methods utilize hybrid formation when single-strandedDNAs or RNAs are complementary to each other. These methods comprise thesteps of: separating unknown nucleic acid fragments according to sizeby, for example, agarose electrophoresis; converting the nucleic acidfragments in the gel to single strands by, for example, alkalitreatment; transferring the single strands to a filter to beimmobilized; and hybridizing the immobilized single-stranded nucleicacid to the labeled probe.

In the case of using, for example, alkaline phosphatase as a marker,when a chemiluminescent substrate, e.g., a 1,2-dioxycetane compound(PPD) is reacted with nucleic acids in contact with a labeled probe in afilter to detect the marker, only the hybridized nucleic acids emitlight. By exposing X-ray films to this light, the size andelectrophoresis position of the target nucleic acid can be confirmed.

(VI) Nucleotide Sequencing Method for Nucleic Acid Molecules

The nucleotide sequencing method of the invention for nucleic acidmolecules comprises the steps of:

-   (a) mixing a template nucleic acid, the DNA synthesizing composition    of the invention. (DNA synthesizing composition comprising the    composition for enhancing synthesis of DNA of the invention and an    enzyme having DNA polymerase activity), nucleotides or nucleotide    derivatives, primers and a release factor, to form a mixture;-   (b) incubating the mixture under such conditions that DNA is    synthesized at a rate of at least 30 bases/second and the error rate    in PCR when using pMol 21 as a template is 4% or less, to amplify a    nucleic acid molecule complementary to the entire or part of the    target nucleic acid to be sequenced; and-   (e) separating the amplified nucleic acid molecule to determine the    entire or partial nucleotide sequence.

In step (b) of the above method, it is preferable to use conditions suchthat the error rate in PCR when using pMol 21 as a template is 1% orless. In addition, it is preferable to use conditions such that 20 kb ormore of target DNA in the template nucleic acid is amplified.

In the above sequencing method, the nucleic acid to be sequenced ispreferably a purified one. The nucleic acid purification method is asdescribed above in the DNA synthesis method of the invention.

The DNA amplification method of the invention may further comprise,between steps (b) and (e), (or between steps (b) and (d) when the methodfurther comprises step (d)), the step of (c) incubating a mixturecontaining a first nucleic acid molecule under such conditions that DNAis synthesized at a rate of at least 30 bases/second and the error ratein PCR when using pMol 21 as a template is 4% or less, to prepare asecond nucleic acid molecule complementary to the entire or part of thefirst nucleic acid molecule.

The DNA amplification method of the invention may further comprise,between steps (b) and (e) (or between steps (c) and (e) when the methodfurther comprises step (c)), the step of (d) purifying the amplifiednucleic acid molecule. The purification method is also as describedabove in the DNA synthesis method of the invention.

In the sequencing method of the invention, the DNA synthesizingcomposition of the invention may be added according to a hot start PCRmethod.

The Maxam-Gilbert method (Maxam, A. M. and Gilbert, W., Proc. Natl.Acad. Sci. USA 74: 560-564, 1997) and the Sanger method (Sanger, F. andCoulson, A. R., J. Mol. Biol. 94: 444-448, 1975) are widely knownnucleotide sequencing methods for nucleic acids.

In the Maxam-Gilbert method, the target DNA is radiolabeled and dividedinto 4 samples. Specific nucleotide bases in DNA are selectivelydestroyed and the samples are treated with chemical reagents that cleavethe molecule at the lesion site. The obtained fragments are separatedinto different bands by gel electrophoresis and an x-ray film is exposedto the gel so that the sequence of the original DNA molecule can be readoff the film.

In contrast, the Sanger method utilizes the DNA synthesis activity of aDNA polymerase. In this method, the nucleic acid to be sequenced and DNApolymerase are mixed with a mixture of a reaction terminatordideoxynucleotide triphosphate (Sanger, F. et al., Proc. Natl. Acad.Sci. USA 74: 5463-5467, 1977) and a short primer (either of which can belabeled in a detectable manner) to synthesize a series of new DNAfragments that are specifically terminated at one of the four dideoxybases. Subsequently, these fragments are analyzed by gel electrophoresisto determine their sequences. By performing four different reactions(one for each ddNTP), considerably complicated DNA molecule sequencescan be quickly determined (Sanger, F. et al., Nature 265: 678-695, 1977;Barnes, W., Meth. Enzymol. 152: 538-556, 1987).

(VII) DNA Molecule Synthesis Kit

The DNA molecule synthesis kit of the invention comprises one or morekinds of constituent compounds or components of the DNA synthesizingcomposition of the invention.

The kit may be composed of two or more separate parts or all theconstituent components may be entirely contained in one part. When thekit is composed of two or more parts, the constituent compounds orcomponents may be separately contained in any of the parts.

The kit of the invention may contain, in addition to a DNA polymerase,various components that can be contained in the above reactioncomposition.

(VIII) DNA Polymerase-Related Factor

The first DNA polymerase-related factor of the invention is aThermococcus species-derived thermostable DNA polymerase-related factorcapable of promoting the DNA synthesis activity of DNA polymerase.

The second DNA polymerase-related factor of the invention is aThermococcus species-derived thermostable DNA polymerase-related factorcapable of binding to DNA polymerase.

Definition

In the specification, “DNA polymerase-related factor” refers to a factorthat affects the functions of DNA polymerase when used with a DNApolymerase. Specific examples include factors capable of promoting theDNA synthesis activity of DNA polymerase, factors capable of binding topolymerase, and factors having both functions or activities.

The method for determining the activity of promoting the DNA synthesisactivity of DNA polymerase is not particularly limited and may be anymethod typically used for determining the DNA synthesis activity of DNApolymerase. More specifically, “promotion of the DNA synthesis activityof DNA polymerase” means an action of enhancing any of processability,DNA elongation rate and dNTP incorporation activity (preferably anincrease of 50% or more, particularly 100% or more)

Processability

Processability refers to the number of nucleotides synthesized duringthe period from the binding of DNA polymerase to the substrate DNA toits release.

One example of a processability determination method is shown below. Areaction solution containing DNA polymerase (120 mM Tris-HCl buffersolution (pH 8.0), 1 mM magnesium chloride, 10 mM KCl, 6 mM (NH₄)₂SO₄,0.1% TritonX-100, 10 μg/ml BSA, 0.2 mM dNTP) is reacted with substrateDNA consisting of single-stranded M13mp18 DNA to which has been annealedprimers labeled with ³²P at the 5′-end at 75° C. under such conditionsthat the substrate is present in a several-fold molar excess relative tothe DNA polymerase. By using such conditions under which the turnover ofDNA polymerase is unlikely to happen, the number of nucleotidessynthesized without release of DNA polymerase from the substrate DNA canbe determined. After a certain reaction time, the reaction is terminatedby adding a reaction terminating solution (50 mM sodium hydroxide, 10 mMEDTA, 5% Ficoll, 0.05% Bromophenol Blue) in a volume equal to thereaction mixture.

The DNAs synthesized by the above reaction are fractionated byelectrophoresis on an alkaline agarose gel, and the gel is dried andsubjected to autoradiography. Labeled λ/HindIII is used as a DNA sizemarker. Processability is determined by measuring the number ofsynthesized nucleotides with reference to the marker band.

DNA Elongation Rate

“DNA elongation rate” refers to the number of DNA molecules synthesizedper unit time. The DNA elongation rate can be determined in thefollowing manner. A reaction solution containing DNA polymerase (120 mMTris-HCl buffer solution (pH 8.0), 1 mM magnesium chloride, 10 mM KCl, 6mM (NH₄)₂SO₄, 0.1% TritonX-100, 10 μg/ml BSA, 0.2 mM dNTP, 0.2 μCi[α-³²P]dCTP) is reacted at 75° C. with single-stranded M13 mp18 DNA towhich primers has been annealed. After a certain reaction time, thereaction is terminated by adding an equal volume of a reactionterminating solution (50 mM sodium hydroxide, 10 mM EDTA, 5% Ficoll,0.05% Bromophenol Blue).

The DNA synthesized by the above reaction is fractionated byelectrophoresis on an alkaline agarose gel, and the gel is dried andsubjected to autoradiography. Labeled λ/HindIII is used as a DNA sizemarker. The DNA elongation rate is determined by measuring thesynthesized DNA size using the marker band as an index.

dNTP Incorporation Activity

“dNTP incorporation activity” refers to a catalytic activity tointroduce deoxyribonucleotide-5′-monophosphate, according to a template,into deoxyribonucleic acid by covalently binding α-phosphate ofdeoxyribonucleotide-5′-triphosphate to the 3′-hydroxyl group of anoligonucleotide or polynucleotide annealed to the template DNA.

If the enzymatic activity of a sample is high, activity measurementshall be carried out after the sample is diluted with a stock buffer(for example, 50 mM Tris-HCl (pH8.0), 50 mM KCl, 1 mM dithiothreitol,0.1% Tween 20, 0.1% Nonidet P40, 50% glycerin).

According to the invention, 25 μl of Solution A, 5 μl of Solution B and5 μl of Solution C shown below, 10 μl of sterilized water and 5 μl of anenzyme solution are pipetted into a microtube and reacted at 75° C. for10 minutes. The reaction solution is then ice-cooled, and 50 μl ofSolution E and 100 μl of Solution D are added and stirred, followed byice-cooling for 10 minutes. The solution is filtered through a glassfilter (Wattman GF/C Filter), and the filter is well washed withSolution D and ethanol, and the radioactivity of the filter is countedin a liquid scintillation counter (Packard) to determine theincorporation of the nucleotide into the template DNA. 1 unit of enzymeactivity is defined as the amount of enzyme that catalyzes theincorporation of 10 nmol of nucleotides into an acid-insoluble fraction(i.e., DNA fraction which becomes insoluble when Solution D is added)per 30 minutes under the above conditions.

Solution A: 40 mM Tris-HCl buffer (pH 7.5)

-   -   16 mM magnesium chloride    -   15 mM dithiothreitol    -   100 μg/ml BSA        Solution B: 2 μg/μl activated calf thymus DNA        Solution C: 1.5 mM dNTP (250 cpm/pmol [³H]dTTP)        Solution D: 20% trichloroacetic acid (2 mM sodium pyrophosphate)        Solution E: 1 mg/ml salmon sperm DNA        Properties

The DNA polymerase-related factor of the invention is a thermostableprotein. Therefore, the factor can be used in DNA synthesis reactionsperformed under conditions of high temperature using a thermostable DNApolymerase.

In this specification, “thermostable” means retaining a capability topromote DNA synthesis activity even after heat treatment. Morespecifically, it means that when 2 nM or more of the DNApolymerase-related factor of the invention is contained with 6 nM of aDNA polymerase in a 20 mM Tris-HCL (pH 7.5 at 75° C.) buffer solution,at least 50%, preferably 75% or more, and particularly preferably 90% ormore of its capability to promote DNA synthesis activity is retainedafter heat treatment at 80° C. for 15 minutes, as compared to theuntreated case.

DNA Polymerase-Related Factor Promoting the DNA Synthesis Activity ofDNA Polymerase (First DNA Polymerase-Related Factor of the Invention)

Examples of DNA polymerase-related factors promoting the DNA synthesisactivity of DNA polymerase include thermostable DNA polymerase-relatedfactors derived from Thermococcus species. In particular, DNApolymerases from the hyperthermophilic archaeon Thermococcuskodakaraensis (KOD DNA polymerase) are preferable and DNApolymerase-related factors from the Thermococcus kodakaraensis KOD1 areparticularly preferable.

Such DNA polymerases whose activities are promoted by the DNApolymerase-related factor of the invention are not particularly limitedand examples include DNA polymerases such as pol I derived from E. coli,and thermostable DNA polymerases such as Tth DNA polymerase derived fromThermus thermophilus, Taq DNA polymerase derived from Thermus aquaticus,Pfu DNA polymerase derived from Pyrococcus furiosus, and DNA polymerasederived from Thermococcus kodakaraensis (KOD DNA polymerase).Thermostable DNA polymerases, in particular, DNA polymerases derivedfrom hyperthermophilic archaeon are preferable. Specific examplesinclude DNA polymerases from the hyperthermophilic archaeon Thermococcuskodakaraensis (KOD DNA polymerase).

One example of a KOD DNA polymerase is an enzyme comprising a DNApolymerase-constituent protein having the amino acid sequence of SEQ IDNO: 1.

The DNA polymerase-related factor of the invention may be capable ofpromoting the activity of merely a specific DNA polymerase butpreferably is capable of promoting the activities of several kinds ofDNA polymerases derived from various sources.

Furthermore, the combined use of two or more DNA polymerase-relatedfactors of the invention enables coexisting DNA polymerases to exhibitfurther enhanced DNA polymerase activity as compared with a single use.

DNA Polymerase-Related Factor Capable of Binding to DNA Polymerase(Second DNA Polymerase-Related Factor of the Invention)

Examples of DNA polymerase-related factors capable of binding to DNApolymerase include thermostable DNA polymerase-related factors derivedfrom Thermococcus species. In particular, DNA polymerases derived fromthe hyperthermophilic archaeon Thermococcus kodakaraensis (KOD DNApolymerase) are preferable and DNA polymerase-related factors from theThermococcus kodakaraensis KOD1 are particularly preferable.

In this specification, the DNA polymerase-related factors capable ofbinding to DNA polymerase includes not only those that can directly bindto DNA polymerase but also those that can bind to DNA polymeraseindirectly via other substances such as other DNA polymerase-relatedfactors.

The DNA polymerase to which the DNA polymerase-related factor of theinvention is bound is not particularly limited and examples include DNApolymerases such as pol I derived from E. coli, and thermostable DNApolymerases such as Tth DNA polymerase derived from Thermusthermophilus, Taq DNA polymerase derived from Thermus aquaticus, Pfu DNApolymerase derived from Pyrococcus furiosus, and DNA polymerase derivedfrom Thermococcus kodakaraensis (KOD DNA polymerase). Thermostable DNApolymerases, in particular, DNA polymerases derived fromhyperthermophilic archaea are preferable. Specific examples include DNApolymerases from the hyperthermophilic archaeon Thermococcuskodakaraensis (KOD DNA polymerase).

The DNA polymerase-related factor of the invention may be capable ofbinding to a specific DNA polymerase or capable of binding to severalkinds of DNA polymerases derived from various sources. The latter ispreferable.

Methods usable for measuring the binding of the DNA polymerase-relatedfactor to DNA polymerase include, for example, a method comprisingmixing the DNA polymerase-related factor and DNA polymerase, followed bynondenatured gel electrophoresis and gel filtration to ascertain thechange in molecular weight, a method of determining the adsorption ofthe DNA polymerase-related factor to a DNA polymerase-immobilizedcarrier, etc.

Amino Acid Sequence and DNA Sequence

Examples of DNA polymerase-related factors of the invention includethose comprising any one of the proteins (e) to (j) below:

-   (e) a protein comprising the amino acid sequence of SEQ ID NO: 2;-   (f) a protein which comprises an amino acid sequence resulting from    addition, deletion or substitution of one or more amino acids in the    sequence of SEQ ID NO: 2, and which can promote the DNA synthesis    activity of a DNA polymerase or bind to a DNA polymerase;-   (g) a protein comprising the amino acid sequence of SEQ ID NO: 4;-   (h) a protein which comprises an amino acid sequence resulting from    addition, deletion or substitution of one or more amino acids in the    sequence of SEQ ID NO: 4, and which can promote the DNA synthesis    activity of a DNA polymerase or bind to a DNA polymerase;-   (i) a protein comprising the amino acid sequence of SEQ ID NO: 6;-   (j) a protein which comprises an amino acid sequence resulting from    addition, deletion or substitution of one or more amino acids in the    sequence of SEQ ID NO: 6, and which can promote the DNA synthesis    activity of a DNA polymerase or bind to a DNA polymerase.

Proteins (f), (h) and (j) are functional equivalents to proteins (e),(g), and (i) respectively. The term “functional equivalents” refers tothose which are substantially equivalent in their functions andactivities even though they are structurally different. The functionalequivalents are included in the scope of the DNA polymerase-relatedfactors of the invention.

Proteins comprising a part of the amino acid sequence of SEQ ID NO: 2, 4or 6 are also included in the scope of the DNA polymerase-relatedfactors of the invention.

The term “proteins comprising” as used herein refers to the proteinsdescribed below, which are also included in the present invention. Thatis, when a protein is produced by genetic engineering techniques, it isoften expressed as a fusion protein. For instance, in order to increasethe target protein expression level, an N-terminal peptide chain derivedfrom other proteins may be added to the N-terminus; or, with the purposeof facilitating the purification of the target protein, the protein maybe expressed by adding an appropriate peptide chain at the N-terminus orC-terminus of the target protein, and a carrier having affinity with thepeptide chain may be used. Such fusion proteins are also included in theDNA polymerase-related factors of the invention.

Among the proteins (e), the protein consisting of the amino acidsequence of SEQ ID NO: 2 is preferable. Among the proteins (f),preferable are those consisting of an amino acid sequence resulting fromaddition, deletion or substitution of one or more amino acids in thesequence of SEQ ID NO: 2, and are capable of promoting the DNA synthesisactivity of a DNA polymerase or binding to a DNA polymerase. Among theproteins (g), the protein consisting of the amino acid sequence of SEQID NO: 4 is preferable. Among the proteins (h), preferable are thoseconsisting of an amino acid sequence resulting from addition, deletionor substitution of one or more amino acids in the sequence of SEQ ID NO:4, and are capable of promoting the DNA synthesis activity of a DNApolymerase or binding to a DNA polymerase. Among the proteins (i), theprotein consisting of the amino acid sequence of SEQ ID NO: 6 ispreferable. Among the proteins (j), preferable are those consisting ofan amino acid sequence resulting from addition, deletion or substitutionof one or more amino acids in the sequence of SEQ ID NO: 6, and arecapable of promoting the DNA synthesis activity of a DNA polymerase orbinding to a DNA polymerase.

Proteins consisting of the amino acid sequence of SEQ ID NO: 2 isKOD-PCNA (proliferating cell nuclear antigen) derived from theThermococcus kodakaraensis KOD1. Proteins consisting of the amino acidsequence of SEQ ID NO: 4 is KOD-RFCS (replication factor C smallsubunit) derived from the Thermococcus kodakaraensis KOD1. Proteinsconsisting of the amino acid sequence of SEQ ID NO: 6 is KOD-RFCL(replication factor C large subunit) derived from the Thermococcuskodakaraensis KOD1.

Genes Encoding the DNA Polymerase-Related Factors of the Invention

The genes encoding the DNA polymerase-related factors of the inventionare those encoding the above proteins (e) to (j). Examples of such genesinclude the following genes (k) to (p):

-   (k) a gene comprising the nucleotide sequence of SEQ ID NO: 3;-   (l) a gene which hybridizes with the gene comprising the nucleotide    sequence of SEQ ID NO: 3 under stringent conditions, and which can    promote the DNA synthesis activity of a DNA polymerase or bind to a    DNA polymerase;-   (m) a gene comprising the nucleotide sequence of SEQ ID NO: 5;-   (n) a gene which hybridizes with the gene comprising the nucleotide    sequence of SEQ ID NO: 5 under stringent conditions, and which can    promote the DNA synthesis activity of a DNA polymerase or bind to a    DNA polymerase;-   (o) a gene comprising the nucleotide sequence of SEQ ID NO: 7;-   (p) a gene which hybridizes with the gene comprising the nucleotide    sequence of SEQ ID NO: 7 under stringent conditions, and which can    promote the DNA synthesis activity of a DNA polymerase or bind to a    DNA polymerase.

The term “gene” used herein includes DNA and RNA. DNAs or RNAs having asequence complementary to the nucleotide sequences described herein arealso included in the scope of the gene of the invention. Furthermore,double-stranded nucleic acids are also included in the scope of the geneof the invention.

A “gene that hybridizes to a specific gene” used herein has a nucleotidesequence similar to that of the specific gene. There is a highpossibility that the amino acid sequence and functions of the proteinencoded by such a gene are also similar to those of the protein encodedby the specific gene.

The homology of the nucleotide sequence of the genes can be determinedby checking whether or not the DNAs or RNAs of the two genes or partialstrands thereof can hybridize to each other under stringent conditions.By using this method, genes that encode proteins having functionssimilar to those of the protein encoded by a specific gene can beobtained.

Herein, “stringent conditions” refers to conditions in which nonspecifichybridization does not occur. More specifically, for example, thefollowing conditions can be mentioned. A DNA-immobilized membrane isreacted with a labeled DNA probe at 50 for 12 to 20 hours in 6×SSC(1×SSC: 0.15 M NaCl, 0.015 M sodium citrate, pH 7.0) containing 0.5%SDS, 0.1% bovine serum albumin (BSA), 0.1% polyvinyl pyrrolidone, 0.1%Ficol 400, and 0.01% denatured salmon sperm DNA. After completion of thereaction, the membrane is washed at a temperature of 3° C. to 50° C. in2× to 0.1×SSC containing 0.5% SDS, until the immobilized labeled DNAprobe signal can be distinguished from the background. Such conditionsare referred to as stringent conditions herein and any gene thathybridizes with a gene comprising the nucleotide sequence of SEQ ID NO:3, 5 or 7 under such conditions and is capable of promoting the DNAsynthesis polymerase activity of DNA polymerase or binding to DNApolymerase is included in the scope of the invention.

Among the genes (k), the gene consisting of the nucleotide sequence ofSEQ ID NO: 3 is preferable. Among the genes (1), preferable are thosethat hybridize with the gene consisting of the nucleotide sequence ofSEQ ID NO: 3 under stringent conditions and that are capable ofpromoting the DNA synthesis activity of a DNA polymerase or binding to aDNA polymerase. Among the genes (m), the gene consisting of thenucleotide sequence of SEQ ID NO: 5 is preferable. Among the genes (n),preferable are those that hybridize with the gene consisting of thenucleotide sequence of SEQ ID NO: 5 under stringent conditions and thatare capable of promoting the DNA synthesis activity of a DNA polymeraseor binding to a DNA polymerase. Among the genes (o), the gene consistingof the nucleotide sequence of SEQ ID NO: 7 is preferable. Among thegenes (p), preferable are those that hybridize with the gene consistingof the nucleotide sequence of SEQ ID NO: 7 under stringent conditionsand that are capable of promoting the DNA synthesis activity of a DNApolymerase or binding to a DNA polymerase.

Cloning of the Gene Encoding the DNA Polymerase-Related Factor of theInvention

The gene encoding the DNA polymerase-related factor of the invention canbe obtained, for example, by the following method:

It has been reported that PCNA (proliferating cell nuclear antigen;hereinafter referred to as “PCNA”) forms a complex with RFC (replicationfactor C; hereinafter referred to as “RFC”) and plays a role in DNAreplication [Seikagaku (Biochemistry), volume 8, (1996), 1542-1548].Therefore, it was expected that in Thermococcus kodakaraensis also,proteins corresponding to PCNA and RFC are expressed and play a role inDNA synthesis reactions. The genes encoding PCNA and RFC homologues ofThermococcus kodakaraensis were obtained by the steps described below.

The entire nucleotide sequences of chromosomal DNAs of the archaeaMethanococcus jannaschii and Pyrococcus horikoshii have been alreadyelucidated, and the nucleotide sequences were presumed to contain genesthat encode proteins deduced to be homologues of PCNA and the RFC smalland large subunits. Genes encoding the homologues of PCNA, RFC smallsubunit (hereinafter also referred to as “RFCS”) and large subunit(hereinafter also referred to as “RFCL”) of these species and thecorresponding known genes were compared to each other to search forhighly homologous nucleotide sequences. By reference to the searchresults, pairs of primers used to obtain gene fragments encoding PCNA,RFC small subunit (RFCS) and large subunit (RFCL) were designed.

By using each pair of primers to obtain PCNA, RFC small subunit (RFCS)and large subunit (RFCL), PCR was performed on template chromosomal DNAof the hyperthermophilic archaeon Thermococcus kodakaraensis KOD1 with aKOD-derived DNA polymerase, “KOD-Plus” (product of Toyobo Co., Ltd.) toamplify DNA fragments. The DNA sequences of the amplified fragments weredetermined and the acquisition of the target genes (KOD-PCNA, KOD-RFCLand KOD-RFCS genes) was confirmed by homology comparison with knowngenes.

Subsequently, using these fragments as probes, a phage DNA librarycontaining the chromosomal DNA of KOD1 prepared by a partial restrictionenzyme digestion was subjected to plaque hybridization to give phageclones PCNA/λ, RFCS/λ and RFCL/λ.

It was found that the cloned KOD-PCNA gene (SEQ ID NO: 3) consists of750 bases, encoding 249 amino acids; the KOD-RFCL gene (SEQ ID NO: 5)consists of 1500 bases, encoding 499 amino acids; and the KOD-RFCS gene(SEQ ID NO: 7) consists of 2601 bases, encoding 866 amino acids.

It was found that the KOD-RFCS gene is cleaved in the RFCS-conservedregion III and an intervening sequence (intein) consisting of 1620 bases(539 amino acids) is present in this portion. Therefore, the interveningsequence was deleted by the PCR fusion method, thus giving a matureKOD-RFCS gene comprising 981 bases (326 amino acids).

(IX) Process for Preparing a DNA Polymerase-Related Factor

The process for preparing a DNA polymerase-related factor of theinvention comprises the step of culturing a transformant harboring thegene of the invention and the step of recovering from the culture athermostable DNA polymerase-related factor which can promote the DNAsynthesis activity of a DNA polymerase, or bind to DNA polymerase, orhave both functions or activities.

In the culture recovery step, the DNA polymerase may be simply recoveredor can be further subjected to purification. Any generally employedmethod for protein purification can be used as the purification method.

For example, a DNA encoding the DNA polymerase-related factor of theinvention (more specifically, for example, a KOD-PCNA, KOD-RFCL orKOD-RFCS gene obtained by the above method) is ligated to an expressionvector, so that the gene can be overexpressed under the control by anexpression vector promoter. In addition, the DNA polymerase-relatedfactor of the invention can be easily recovered from a transformantharboring the gene of the invention by ligating a DNA encoding the DNApolymerase-related factor of the invention to a DNA encoding a histidinetag so as to be expressed as a fusion protein. This is because thefusion protein can be easily isolated by using a usually used nickelcolumn.

(X) DNA Synthesis Method Using the DNA Polymerase-Associated Factor ofthe Invention

The second DNA synthesis method of the invention is a method ofsynthesizing DNA using a DNA polymerase in the presence of the DNApolymerase-related factor of the present invention (comprising one ormore of the proteins (e) to (j)). When a polymerase chain reaction (PCR)is performed by the second DNA synthesis method of the invention, PCRsensitivity can be increased by at least several-fold to severaltens-fold by synthesizing DNA in the presence of the DNA polymerase ofthe invention, as compared to the case of its absence.

In the second DNA synthesis method of the invention, a single DNApolymerase-related factor or a mixture of two or more may be used. Themixed use of two or more is preferable.

Examples of DNA polymerase-related factors usable in the second DNAsynthesis reaction of the invention include KOD-PCNA, KOD-RFCS andKOD-RFCL. For example, when using KOD-PCNA, the DNA elongation rate canbe increased several fold, compared to a reaction with single use of KODDNA polymerase. The above three types of DNA polymerases can be usedsingly or as a KOD-RFC complex comprising KOD-RFCS and KOD-RFCL(heteropentamer of RFCS:RFCL=1:4) or a PCNA-RFC complex comprisingKOD-PCNA and KOD-RFC.

Examples of DNA polymerases usable in the second DNA synthesis method ofthe invention include DNA polymerases such as pol I derived from E.coli; and thermostable DNA polymerases such as Tth DNA polymerasederived from Thermus thermophilus, Taq DNA polymerase derived fromThermus aquaticus, Pfu DNA polymerase derived from Pyrococcus furiosus,and DNA polymerase derived from Thermococcus kodakaraensis (KOD DNApolymerase). Thermostable DNA polymerases are preferable. In particular,DNA polymerases derived from hyperthermophilic archaea, morespecifically DNA polymerases from the hyperthermophilic archaeonThermococcus kodakaraensis (KOD DNA polymerase) are preferable. WhenThermococcus kodakaraensis polymerase and the DNA polymerase-relatedfactor of the invention are used in combination, the origin of the DNApolymerase and the DNA polymerase-related factor is the same so that notonly a high DNA elongation rate and excellent processability but alsohigh DNA synthesis fidelity, which is a characteristic of KOD DNApolymerase, can be obtained, so that particularly excellent effects canbe expected. In the DNA synthesis method of the invention, single DNApolymerases or a mixture of two or more can be used.

DNA can be synthesized by PCR methods using the second DNA synthesismethod of the invention. For example, a method of performing PCR hasbeen reported using a DNA polymerase composition prepared by mixing Pfupolymerase having 3′-5′ exonuclease activity, Tli polymerase and thesevariant enzymes as a method for amplifying long chain nucleic acids(Barns, W. M. Proc. Natl. Acad. Sci. USA, 91, (1994) 2216-2220). Amethod of performing PCR using a DNA polymerase composition prepared bymixing Tth polymerase not having 3′-5′ exonuclease activity, Pfupolymerase having 3′-5′ exonuclease activity or Tli polymerase, andThermotaga maritima-derived thermostable DNA polymerase has also beenreported (Japanese Unexamined Patent Publication No. H8 (1996)-38198).

However, although these compositions provide improved amplificationefficiency as compared to the use of one type of DNA polymerase,sufficient amplification efficiency can not achieved because theycomprise two types of DNA polymerases that differ in thermostability andDNA elongation rate.

Therefore, the present inventors carried out extensive research anddeveloped a novel mixed type thermostable DNA polymerase (KOD Dash DNApolymerase) prepared by mixing a 3′-5′ exonuclease activity-deleted KOD(exo-) DNA polymerase and a KOD DNA polymerase (Japanese UnexaminedPatent Publication No. H10 (1998)-42874). This KOD Dash DNA polymeraseappeared to be the optimal form of a mixed type enzyme and furtherimprovement seemed impossible.

However, even in the case of this enzyme, further enhanced PCRperformance was achieved by adding a DNA polymerase-related factor ofthe invention derived from the same origin as the enzyme (see Example15). Mixed enzymes using two types of DNA polymerases also achieveimproved effects with the addition of the DNA polymerase-related factor.When a mixed DNA polymerase derived from the same origin as the DNApolymerase-related factor (here KOD dash), further improved effects areobtained.

In the second DNA synthesis method of the invention, the amount of theDNA polymerase-related factor to be used is not particularly limited.The DNA polymerase-related factor may be used in an amount sufficient topromote the synthesis activity of a DNA polymerase.

(XI) DNA Synthesis Kit Comprising a DNA Polymerase-Related Factor(Second DNA Synthesis Kit)

The DNA synthesis kit comprising the DNA polymerase-related factor ofthe invention is a kit comprising the DNA polymerase-related factor(comprising one or more of the proteins (e) to (j)) of the invention anda DNA polymerase. The kit may comprise one or more other components asnecessary for DNA synthesis. Examples of other components necessary forDNA synthesis include 4 types of nucleotides or nucleotide derivativessuch as dNTP, buffers, salts such as MgCl₂, and additives useful for DNAsynthesis, primers, and the like. These are described above. Suchcomponents may be entirely contained in one part of the kit orseparately contained in two or more parts.

This kit may be used for various reactions in which a DNA polymerase isused. Thus it may be a kit for performing in vitro DNA synthesisreactions, a kit for performing DNA base sequencing, for instance, bythe dideoxy method, a DNA labeling kit or a PCR kit.

DNA polymerase-related factors preferably contained in the kit of theinvention are, for example, KOD-PCNA, KOD-RFCS and KOD-RFCL. In the kitof the invention, the three types of DNA polymerase-related factors maybe mixed singly with KOD polymerase or used as a KOD-RFC complexcomprising KOD-RFCS and KOD-RFCL (heteropentamer of RFCS:RFCL=1:4) or aPCNA-RFC complex comprising KOD-PCNA and KOD-RFC.

Examples of DNA polymerases contained in the kit of the inventioninclude DNA polymerases such as pol I derived from E. coli; andthermostable DNA polymerases such as Tth DNA polymerase derived fromThermus thermophilus, Taq DNA polymerase derived from Thermus aquaticus,Pfu DNA polymerase derived from Pyrococcus furiosus, and DNA polymerasederived from Thermococcus kodakaraensis (KOD DNA polymerase). In the kitof the invention, thermostable DNA polymerases are preferable.Especially preferable are DNA polymerases derived from hyperthermophilicarchaea. Specific examples thereof include DNA polymerases fromThermococcus kodakaraensis (KOD DNA polymerase).

By performing the above DNA synthesis method using the kit of theinvention, DNA can be synthesized more quickly in a more convenient andhighly sensitive manner.

EXAMPLES

The present invention is described in more detail in the Examples below.However, it should be understood that the present invention is notlimited to these Examples.

Example 1-1 Investigation of the Effect of Oxalate Ion Addition to MixedType Enzyme (EX-Taq) (FIG. 1)

The effect produced by the addition of oxalate ions was investigated inthe PCR using EX-Taq DNA polymerase. A PCR buffer supplied with EX-Taq(TAKARA) and 0.3 μM of a pair of primers of SEQ ID Nos:8 and 9 and 0.2mM dNTPs were used. PCR was performed by using 1.0 U EX-Taq DNApolymerase and 20 ng of genomic DNA derived from human cell line K562 asa template, with the influence on the PCR result caused by varying theconcentration of added oxalate ion being investigated. The PCRconditions were as follows: 1 preliminary reaction of 94° C. for 2minutes, and then 35 cycles of 94° C. for 15 seconds, 60° C. for 30seconds and 68° C. for 8.5 minutes. This PCR amplification was performedby using GeneAmp2400 (PE Applied Biosystems).

FIG. 1 shows the electrophoresis pattern of the PCR product. It wasfound that the target DNA was not amplified in the absence of oxalateion, but the DNA was amplified when 1 mM oxalate ion was added, and theamount of amplification was the highest when 2-3 mM oxalate ion wasadded. The amplification of the target DNA was confirmed with theaddition of oxalate ion in an amount ranging from 1-4 mM.

These results demonstrate the effect produced by the addition of oxalateion to the mixed type enzyme. The concentration range of oxalate ionwithin which the effect of the addition is found is up to 4 mM, but itis expected that the optimal concentration varies depending on the kindand amplification length of the target DNA, PCR buffer composition, etc.

Example 1-2 Investigation of the Type of Salt which is Effective for PCRUsing Mixed Type Enzyme (EX-Taq)(FIG. 2)

Salts (potassium chloride, potassium acetate, potassium oxalate, sodiumoxalate, potassium sulfate) were investigated for their effectiveness inPCR using EX-Taq DNA polymerase under the same conditions as Example 1.Each salts was used in an amount of 4 to 6 mM of ion equivalent

FIG. 2 shows the electrophoresis patterns of the PCR products.Amplification was observed only when potassium oxalate or sodium oxalatewas added as a salt. Amplification was not observed when other presentlywidely used potassium salts were added. These results suggest that theoxalate anions are effective for PCR.

Example 1-3 Investigation of the Type of Carboxylate Salt Effective forPCR Using Mixed Type Enzyme (EX-Taq)(FIG. 3)

The type of carboxylate salts (potassium oxalate, sodium oxalate,potassium succinate, potassium formate) that is effective for PCR usingEX-Taq DNA polymerase was investigated under the same conditions asExample 2.

FIG. 3 shows the electrophoresis patterns of the PCR products. As inExample 2, amplification of the target DNA was observed only whenpotassium oxalate or sodium oxalate was added as a salt. A similareffect to that produced by oxalate salts was not observed with formatehaving a monovalent carboxyl group, or succinate having a divalentcarboxyl group. However, compared to oxalates, potassium formate andpotassium succinate may have a different concentration range that iseffective for PCR amplification. Therefore, the effectiveness of thesesalts cannot be completely ruled out. It is assumed that in certainconcentration ranges, other carboxylate salts might possibly beeffective for PCR.

Example 1-4 Investigation of the Effect of *Oxalate Ions* Addition toPolI Type Enzyme (Tag)

The effect produced by the addition of oxalate ions was investigated inPCR using Taq DNA polymerase. A PCR buffer (10 mM Tris-HCl (pH 8.3), 50mM KCl, 1.5 mM MgCl₂) attached to Taq (TOYOBO CO., LTD.), 0.2 mM dNTPsand 0.3 μM of a primer pair were used. A pair of primers of SEQ IDNos:10 and 11 was used for the PCR amplification of a 3.6-kb DNA ofβ-globin cluster (the electrophoresis pattern of its PCR product isshown in FIG. 4), while a pair of primers of SEQ ID Nos:12 and 13 wasused for the PCR amplification of a 4.5-kb DNA of myelin oligodendrocyteglycoprotein (the electrophoresis pattern of its PCR product is shown inFIG. 5). The influence of varying the concentration of oxalate ionsadded on the PCR results was investigated using 2.5 U Taq DNA polymeraseand 20 ng of Genomic DNA derived from cultured human cell K562 as atemplate. The PCR conditions were as follows: 94° C. for 2 minutes as 1preliminary reaction, and then 30 cycles of 94° C. for 15 seconds, 60°C. for 30 seconds and 68° C. for 8 minutes. The PCR amplification wasperformed by using GeneAmp 2400 (PE Applied Biosystems).

When no oxalate ions were added, the electrophoresis patterns of all thetarget sequences showed only a smear. However, in the PCR amplificationof 3.6-kb DNA of β-globin, when 2 mM oxalate ion was added,amplification of the target sequence was observed together with a smear,and when 4 mM oxalate ion was added, no smear was observed. With theaddition of 4-5 mM oxalate ion, a discrete amplification band wasobserved. In the PCR amplification of 4.5-kb DNA of myelinoligodendrocyte glycoprotein electrophoresis patterns, amplification ofthe target DNA was observed together with a smear when 1 mM oxalate ionwas added, and when 2 mM oxalate ion was added, no smear was observed. Adiscrete amplification band of the target DNA was confirmed with theaddition of 2-5 mM oxalate ion.

These results demonstrate the effect produced by the addition of oxalateion to the Pol I enzyme. The concentration range within which the effectof the addition of oxalate ion is found was up to 5 mM, but it isexpected that the optimal concentration varies depending on the kind andamplification length of the target DNA, PCR buffer composition, etc.

Example 1-5 Investigation of the Effect of Oxalate Ion Addition toα-Type Enzyme (Native Pfu)(FIG. 6)

The effect produced by the addition of oxalate ion in PCR wasinvestigated using nPfu polymerase. A PCR buffer (20 mM Tris-HCl (pH8.75), 10 mM KCl, 10 mM (NH₄) ₂SO₄, 2 mM MgSO₄, 0.1% Triton X-100, 100μg/ml BSA) attached to nPfu (STRATAGENE), 0.2 mM of dNTPs, and 0.3 μM ofa pair of primers of SEQ ID NOs:12, and 13 were used. PCR was performedby using 2.5 U nPfu DNA polymerase and 20 ng of genomic DNA derived fromhuman cell line K562 as a template, with the influence on the PCR resultcaused by varying the concentration of oxalate ion being investigated.PCR was performed by using GeneAmp2400 (PE Applied Biosystems) with thefollowing conditions: 1 preliminary reaction of 94° C. for 2 minutes,and then 35 cycles of 94° C. for 15 seconds, 60° C. for 30 seconds and68° C. for 8 minutes.

The electrophoresis patterns of the PCR products are shown in FIG. 6.When no oxalate ions were added, only a smear was shown. However, when2-5 mM oxalate ion was added, amplification of the target DNA wasobserved. These results demonstrate the effect produced by the additionof oxalate ion on the α-type enzyme. The concentration range withinwhich the effect of the addition of oxalate ion is found isapproximately up to 5 mM, but it is expected that the optimalconcentration varies depending on the kind and size of amplified portionof the target DNA, PCR buffer composition, etc.

Example 1-6 Investigation of the Type of Salt which is Effective for PCRUsing α-Type Enzyme (Native Pfu)(FIG. 7)

Salts (potassium chloride, potassium acetate, potassium oxalate,potassium sulfate) were tested for their effectiveness in PCR using nPfuDNA polymerase under the same conditions as Example 1-5. The amount ofeach salt used was 4-6 mM of ion equivalent, as was the most effectivein Example 1-5.

The electrophoresis patterns of the PCR products are shown in FIG. 7.When potassium salts (potassium chloride, potassium acetate, potassiumoxalate, potassium sulfate), which are widely used today, were added,amplification of the target DNA was not observed, and only whenpotassium oxalate was added, was amplification of the target DNAobserved. These results demonstrate that oxalate anions are effectivefor PCR.

Example 1-7 Investigation of the Effect Produced by the Addition ofOxalate Ion to Hot Start Enzyme (KOD-plus)(FIG. 8)

The effect produced by the addition of oxalate ion to a hot start enzymewas investigated using a hot start enzyme KOD-plus DNA polymerasecontaining KOD α-type enzyme) and two kinds of KOD antibodies. The PCRbuffer supplied with KOD-plus (manufactured by Toyobo), 0.2 mM dNTPs and0.3 μM of a primer pair of SEQ ID Nos:8 and 9 were used. PCR wasperformed using 2.5 U KOD-plus DNA polymerase and 20 ng of Genomic DNAderived from human cell line K562 as a template to investigate theinfluence of varying the concentration of oxalate ion added on the PCRresults. The PCR conditions were as follows: 1 preliminary reaction of94° C. for 2 minutes, and then 35 cycles of 94° C. for 15 seconds, 60°C. for 30 seconds and 68° C. for 8.5 minutes. The PCR was performedusing GeneAmp2400 (PE Applied Biosystems).

The electrophoresis patterns of the PCR products are shown in FIG. 8. Inthe absence of oxalate ion, many extra bands were observed, but in thepresence of potassium oxalate, no extra bands were observed and only thetarget DNA was amplified. The optimal concentration of potassium oxalatewas about 3 mM. When 5 mM of potassium oxalate was added, a decrease inthe amount of the target DNA amplified was found.

These results demonstrate the effect produced by the addition of oxalateion to the hot start enzyme. The concentration range within which theeffect of the addition of oxalate ion is found is approximately up to 5mM, but it is expected that the optimal concentration varies dependingon the kind and size of amplified portion of the target DNA, PCR buffercomposition, etc.

Example 1-8 Investigation of the Type of Salt which is Effective for PCRUsing Hot Start Enzyme (KOD-plus)(FIG. 9)

Salts (potassium chloride, potassium acetate, potassium formate,potassium oxalate, sodium oxalate, potassium succinate, potassiumsulfate) were tested for their effectiveness in PCR using KOD-plus DNApolymerase under the same conditions as Example 1-7. The amount of eachsalt used was 6 mM of ion equivalent, as was the most effective inExample 1-7.

The electrophoresis patterns of the PCR products are shown in FIG. 9.Amplification of the target DNA was not observed when other presentlywidely used potassium salts (potassium chloride, potassium acetate,potassium oxalate, potassium sulfate) were added. Amplification wasobserved only when potassium oxalate or sodium oxalate was added.

Potassium formate and potassium succinate, whose chemical formulas arerelatively similar to that of oxalate, were also investigated, butsimilar effects to those of oxalate were not found. However, compared tooxalate, potassium formate and potassium succinate may have a differentconcentration range that is effective for PCR amplification. Therefore,the effectiveness of these salts cannot be completely ruled out. It maybe assumed that in certain concentration ranges, other carboxylate saltsmight be effective for PCR.

These results demonstrate the effect produced by the addition of oxalateanions to the hot start enzyme.

Example 1-9 Investigation of the Effect Produced by the Addition ofMalonic Acid Ion and Maleic Acid Ion to Hot Start Enzyme (KOD-plus)(FIG.10)

The type of salts other than oxalates (sodium malonate, sodium maleate)were tested for their effectiveness in PCR under the same conditions asExample 1-7.

The electrophoresis patterns of the PCR products are shown in FIG. 10.When 2 mM of sodium malonate was added, the extra bands tended todecrease, and when 6 mM was added, most of the extra bands disappeared,indicating amplification of only the target DNA was observed.

Similarly, when 4 mM of sodium maleate was added, extra bands tended todecrease, and when 10 mM was added, most of the extra bands disappeared,indicating amplification of only the target DNA.

These results demonstrate the effect produced by the addition ofmalonate and maleate, which are both dicarboxylates. The concentrationranges of these salts within which the effects of the addition are foundwere higher than that of oxalate. It is expected that the optimalconcentration of each of the dicarboxylic acids varies depending on thekind and amplification length of the target DNA, PCR buffer composition,etc.

Example 2-1 Investigation of Synergistic Effects Produced by theAddition of Oxalate Ion and Betaine to Mixed-Type Enzyme (EX-Taq)(FIG.11)

Oxalate ion and betaine were investigated for their synergisticeffectiveness in amplification of a target DNA which cannot be usuallyamplified by PCR using EX-Taq DNA polymerase (TAKARA). Potassium oxalatewas added to the PCR buffer attached to EX-Taq DNA polymerase to attaina final concentration of 2 mM. Also used were 0.2 mM dNTPs, 0.3 μM of apair of primers consisting of the sequences of ID NOs:14 and 15respectively, and 1.0 U EX-Taq DNA polymerase. 20 ng of genomic DNAderived from human cell line K562 was used as a template. This reactionmixture was subjected to PCR by using GeneAmp2400 (PE AppliedBiosystems) to investigate the influence of varying the concentration ofbetaine added on the PCR results. The PCR conditions were as follows:1preliminary reaction of 94° C. for 2 minutes, and then 35 cycles of 94°C. for 15 seconds, 60° C. for 30 seconds and 68° C. for 8.5 minutes.

The electrophoresis patterns of the PCR products are shown in FIG. 11.In the absence of betaine, amplification of the target DNA was barelynoticeable. In the presence of 0.5 M betaine, an increase inamplification amount was observed. These results demonstrate asynergistic effect produced by the addition of oxalate ion and betaineto the mixed-type enzyme.

Example 2-2 Investigation of Synergistic Effects Produced by theAddition of Oxalate Ion and Betaine to α-Type Enzyme (Native Pfu)(FIG.12)

Oxalate ion and betaine were investigated for their synergisticeffectiveness in amplification of a target DNA which cannot be usuallyamplified by PCR using nPfu polymerase. Potassium oxalate was added tothe PCR buffer (20 mM Tris-HCl(pH: 8.75), 10 mM KCl, 10 mM (NH₄)₂SO₄, 2mM MgSO₄₁ 0.1% Triton X-100, 100 μg/ml BSA) supplied with nPfu(STRATAGENE) to attain a final concentration of 2 mM. Also used were 0.2mM dNTPs, 0.3 μM of a pair of primers of SEQ ID Nos:16 and 17, and 2.5 UnPfu DNA polymerase. 20 ng of genomic DNA derived from human cell lineK562 was used as a template. This reaction mixture was subjected to PCRby using GeneAmp2400 (PE Applied Biosystems) to investigate theinfluence of varying the concentration of betaine added on the PCRresults. The PCR conditions were as follows: 1 preliminary cycle of 94°C. for 2 minutes, and then 35 cycles of 94° C. for 15 seconds, 60° C.for 30 seconds and 68° C. for 8.5 minutes.

The electrophoresis patterns of the PCR products are shown in FIG. 12.When no betaine was added, the electrophoresis patterns of the targetDNA showed only a smear. However, when 0.5 mM of betaine was added,amplification of the target DNA was observed. These results demonstratea synergistic effect produced by the addition of oxalate ion and betaineto α-type enzyme.

Example 2-3 Investigation of Synergistic Effects Produced by theAddition of Oxalate Ion and Betaine to Hot Start Enzyme (KOD-plus)(FIG.13)

Oxalate ion and betaine were investigated for their synergisticeffectiveness in amplification of a target DNA which cannot be amplifiedby normal PCR, using KOD-plus DNA polymerase, i.e. a hot start enzymewhich is a mixture of KOD (α-type enzyme) and two kinds of KODantibodies. Potassium oxalate was added to the PCR buffer supplied withKOD-plus (TOYOBO CO., LTD.) to attain a final concentration of 2 mM.Also used were 0.2 mM dNTPs and 0.3 μM of a pair of primers of SEQ IDNOs: 14 and 15. 20 ng of genomic DNA derived from human cell line K562was used as a template. This reaction mixture was subjected to PCR byusing GeneAmp2400 (PE Applied Biosystems) to investigate the influenceof varying the concentration of betaine on the PCR results. The PCRconditions were as follows: 1 preliminary reaction of 94° C. for 2minutes, and then 35 cycles of 94° C. for 15 seconds, 60° C. for 30seconds and 68° C. for 8.5 minutes.

The electrophoresis patterns of the PCR products are shown in FIG. 13.When no betaines were added, amplification of the target DNA was barelynoticeable. However, when 0.5-1.5 M of betaine was added, an increasedamount of amplification of the target DNA was observed. The amount ofamplification was greatest when 1.5 M of betaine was added.

These results demonstrate a synergistic effect produced by the additionof oxalate ion and betaine to the hot start enzyme.

Example 2-4 Investigation of Synergistic Effects of Oxalate Ion and DMSOwith Hot Start Enzyme (KOD-plus)(FIG. 14)

Oxalate ion and DMSO were investigated for their synergisticeffectiveness for hot start enzyme in amplification of a target DNAwhich is difficult to amplify by usual PCR, using a KOD-plus DNApolymerase, i.e., hot start enzyme. Potassium oxalate was added to thePCR buffer supplied with KOD-plus (TOYOBO CO., LTD.) to attain a finalconcentration of 2 mM. Also used were 0.2 mM dNTPs and 0.3 mM of a pairof primers of SEQ ID Nos:18 and 19, and 1.0 U KOD-plus DNA polymerase.0.5 μL of cDNA obtained by reverse transcription of 500 ng of total RNAderived from human cell line K562 with ThermoScript (Invitrogen) wasused as a template. This reaction mixture was subjected to PCR by usingGeneAmp2400 (PE Applied Biosystems) to investigate the influence on thePCR results of varying the concentration of DMSO. The PCR conditionswere as follows: 1 preliminary reaction of 94° C. for 2 minutes, andthen 40 cycles of 94° C. for 15 seconds, 60° C. for 30 seconds and 68°C. for 9 minutes.

The electrophoresis patterns of the PCR products are shown in FIG. 14.In the absence of DMSO, amplification of the target DNA was barelynoticeable. In the presence of DMSO, an increase in amplification amountwas observed. In the presence of 5% DMSO, sufficient amplification wasobserved. These results demonstrate a synergistic effect produced by theaddition of oxalate ions and DMSO to the hot start enzyme.

Example 2-5 Investigation of Synergistic Effects Produced by theAddition of Potassium Oxalate and Betaine on Long PCR Using α-TypeEnzyme (KOD)(FIG. 15)

Oxalate ion and betaine were investigated for their synergisticeffectiveness in long PCR amplification of a target DNA of more than 20kb derived from the human genome using KOD DNA polymerase, successfulamplification of which using α-type enzyme alone has not been reported.Potassium oxalate was added to the PCR buffer supplied with KOD-plus(TOYOBO CO.,LTD.) to attain a final concentration of 2 mM. Also usedwere 0.4 mM dNTPs, 0.3 μM of a pair of primers of SEQ ID Nos:20 and 21,and 2.0 U KOD DNA polymerase. 200 ng of genomic DNA derived from humancell line K562 was used as a template. This reaction mixture wassubjected to PCR by using GeneAmp2400 (PE Applied Biosystems) toinvestigate the influence on the PCR results of varying theconcentration of betaine. The PCR conditions were as follows: 1preliminary reaction of 94° C. for 2 minutes; 5 cycles of 98° C. for 10seconds and 74° C. for 18 minutes; 5 cycles of 98° C. for 10 seconds and72° C. for 18 minutes; 5 cycles of 98° C. for 10 seconds and 70° C. for18 minutes; 25 cycles of 98° C. for 10 second and 68° C. for 18 minutes;and 1 additional reaction of 68° C. for 7 minutes.

The electrophoresis patterns of the PCR products are shown in FIG. 15.When the betaine concentration was 1.2 M or less, amplification of thetarget DNA was not observed, while sufficient amplification was foundwhen 1.4 M or 1.6 M betaine was used. The amount of betaine used is notlimited to the above concentration however, since the optimalconcentration of betaine varies depending on the kind of enzyme used.

Example 2-6 Investigation of Synergistic Effects Produced by theAddition of Betaine and Potassium Oxalate in Long PCR Using α-TypeEnzyme (KOD)(FIG. 16)

Oxalate ion and betaine were investigated for their synergisticeffectiveness in long PCR amplification of a target DNA of more than 20kb derived from the human genome using KOD DNA polymerase, successfulamplification of which using α-type enzyme alone has not been reported.Betaine was added to the PCR buffer supplied with KOD-plus (TOYOBO CO.,LTD.) to attain a final concentration of 1.5 M. Also used were 2.0 U KODDNA polymerase, 0.4 mM dNTPs and 0.3 μM of a pair of primers of SEQ IDNos:22 and 23 respectively for the PCR amplification of 22 kb DNA in tPAgene, and SEQ ID Nos:22 and 24 respectively for the PCR amplification oftPA 24 kb DNA. 200 ng of genomic DNA derived from human cell line K562was used as a template. This reaction mixture was subjected to PCR byusing GeneAmp2400 (PE Applied Biosystems) to investigate the influenceon the PCR results of varying the concentration of potassium oxalate.The PCR conditions were as follows: 1 preliminary reaction of 94° C. for2 minutes; 5 cycles of 98° C. for 10 seconds and 74° C. for 18 minutes;5 cycles of 98° C. for 10 seconds and 72° C. for 18 minutes; 5 cycles of98° C. for 10 seconds and 70° C. for 18 minutes; 25 cycles of 98° C. for10 seconds and 68° C. of 18 minutes; and 1 additional reaction of 68° C.for 7 minutes.

The electrophoresis patterns of the PCR products are shown in FIG. 16.When tPA 22 kb DNA was the target, its amplification was confirmed onlywhen 2 mM potassium oxalate was added. When tPA 24 kb DNA was thetarget, slight amplification was observed, when 1.5 mM or 2.0 mMpotassium oxalate was added. The amount of potassium oxalate used is notlimited to the above concentration however, since the optimalconcentration of potassium oxalate varies depending on the kind ofenzyme used.

Example 2-7 Investigation of the Effect Produced by Hot Start PCR inLong PCR (FIG. 17)

Under the aforementioned conditions in which oxalate ions and betaineare present, the effect of hot start PCR using antibodies in long PCRusing KOD DNA polymerase was investigated. Potassium oxalate and betainewere added to the PCR buffer supplied with KOD-plus (TOYOBO CO.LTD.) toattain a final oxalate concentration of 2 mM and a final betaineconcentration of 1.5 M. Also used were 2.0 U KOD DNA polymerase, 0.4 mMdNTPs and 0.3 μM of a pair of primers of SEQ ID Nos:22 and 25 for PCRamplification of tPA 12 kb DNA; SEQ ID Nos:22 and 26 for PCRamplification of tPA 15 kb DNA; SEQ ID Nos:22 and 27 for PCRamplification of tPA 18 kb DNA; SEQ ID Nos:22 and 23 for PCRamplification of tPA 22 kb DNA; SEQ ID Nos:22 and 24 for PCRamplification of tPA 24 kb DNA; and SEQ ID Nos:28 and 29 for PCRamplification of β-globin 17.5 kb DNA. 200 ng of genomic DNA derivedfrom human cell line K562 was used as a template. This reaction mixturewas subjected to PCR by using GeneAmp24000 (PE Applied Biosystems) toinvestigate the effect of hot start PCR in the presence or absence ofantibodies. The PCR conditions were as follows: 1 preliminary reactionof 94° C. for 2 minutes; 5 cycles of 98° C. for 10 seconds and 74° C.for 18 minutes; 5 cycles of 98° C. for 10 seconds and 72° C. for 18minutes; 5 cycles of 98° C. for 10 seconds and 70° C. for 18 minutes; 25cycles of 98° C. for 10 seconds and 68° C. for 18 minutes; and 1additional reaction of 68° C. for 7 minutes.

The electrophoresis patterns of the PCR products are shown in FIG. 17.Evaluation of the patterns of 6 type target DNAs revealed that the useof antibodies decreased smearing and increased their amplificationamounts in general. In particular, in the amplification of tPA 22 kbtarget DNA, amplification of the target DNA was not observed when noantibodies were used, but amplification was confirmed when antibodieswere present. Although it was expected that hot start PCR would also beeffective for long PCR, there has been no example which demonstratesthis to date. Therefore, this is supposed to be the first such report ofamplification of human genomic target DNA of 20 kb or more.

Example 2-8 Investigation of the Effects of a Reagent Which is Effectivefor PCR of a GC Rich Target DNA for Long PCR (FIG. 18)

The reagent “PCRx” (Invitrogen), which is considered to be effective fortarget DNAs with a high GC content, and betaine were investigated to seewhether they are similarly effective for long PCR. Potassium oxalate wasadded to the buffer supplied with KOD-plus (TOYOBO CO.,LTD.) to attain afinal concentration of 2 mM. Also used were 1.0 U KOD DNA polymerase,0.4 mM dNTPs and 0.3 μM of a pair of primers of SEQ ID Nos:30 and 31 wasused for PCR amplification of tPA 9 kb DNA, and a pair of primers SEQ IDNos:22 and 23 was used for PCR amplification of tPA 12 kb DNA. 200 ng ofgenomic DNA derived from human cell line K562 was used as a template.This reaction mixture was subjected to PCR by using GeneAmp2400 (PEApplied Biosystems) to compare the effects produced by the addition ofbetaine and PCRx. The PCR conditions were as follows: a preliminarycycle of 94° C. for 2 minutes; 5 cycles of 98° C. for 10 seconds and 74°C. for 12 minutes; 5 cycles of 98° C. for 10 seconds and 72° C. for 12minutes; 5 cycles of 98° C. for 10 seconds and 70° C. for 12 minutes; 25cycles of 98° C. for 10 seconds and 68° C. for 12 minutes; and finallyan additional reaction of 68° C. for 7 minutes.

The electrophoresis patterns of the PCR products are shown in FIG. 18.When no additives were used, amplification of both the tPA 9 kb and 12kb target DNAs was barely observed. However, sufficient amplification ofboth target DNAs was observed when 1 M or 1.5 M betaine was added. Incontrast, when PCRx was used in a 1×, 2× or 3× concentration accordingto the instruction manual, no amplification was observed. These resultsdemonstrate that the reagent, which is effective for GC rich targets, isnot always effective for long PCR and that the effectiveness for longPCR is a property specific to betaine. The effects of betaine for longPCR have been confirmed not only with α-type DNA polymerase also withall the heat-resistant DNA polymerases.

Example 3-1 Cloning of the Gene Coding for DNA Polymerase-Related FactorDerived from Hyperthermophilic Archaeon KOD1

Hyperthermophilic archaeon KOD1, isolated from Kodakara Island,Kagoshima, Japan, was cultured at 95° C. and cells were collected.Chromosomal DNA of hyperthermophilic archaeon KOD1 was prepared from theobtained cells in the standard manner. Primer pairs were synthesizedbased on the amino acid sequences of conserved regions of PCNA(proliferating-cell-nuclear-antigen), RFCS (replication C small subunit)and RFCL (replication C large subunit), which are known as DNApolymerase-related factors. The primer pair of PCNA-f1 (SEQ ID NO:32)and PCNA-r1 (SEQ ID NO: 33) was used for PCR amplification of theKOD-PCNA gene. The primer pair of RFCS-f1 (SEQ ID NO:34) and RFCS-r1(SEQ ID NO:35) was used for PCR amplification of the KOD-RFCS gene. Theprimer pair of RFCL-f1 (SEQ ID NO:36) and RFCL-r1 (SEQ ID NO:37) wasused for PCR amplification of the KOD-RFCL gene. PCR was performed byusing these primer pairs and the prepared chromosomal DNA as a template.All the PCR operations hereinafter were carried out by using KOD-Plus(TOYOBO CO.,LTD.) according to the usage examples supplied with theenzyme. The nucleotide sequences of DNA fragments amplified by PCR weredetermined by the dideoxy chain termination method, whereby each DNAfragment was found to have its own conserved sequence (SEQ ID Nos:38, 39and 40 respectively).

By the in vitro packaging method using DNA fragments obtained bydigesting chromosomal DNA of hyperthermophilic archaeon KOD1 by EcoRIand Gigapack Gold (Stratagene), a cosmid having chromosomal DNA ofhyperthermophilic archaeon KOD1 was packed in λ phage particles,constructing a phage DNA library.

Plaque hybridization was performed by using a DNA fragment containingKOD-PCNA as a probe to obtain from the above-mentioned library phageclone PCNA/λ, which is considered to contain a PCNA gene derived fromKOD1. Phage clone RFCS/λ and RFCL/λ, which are considered to containRFCS and RFCL genes, respectively, were obtained in a similar manner.

Cloning method of the polymerase-related factor genes derived fromhyperthermophilic archaeon KOD1 and the expression vector constructionmethod are shown in FIG. 19.

Example 3-2 Determination of Nucleotide Sequences of Clone Fragments

Phage direct PCR was performed using the primer pair of PCNA-f1 andPCNA-r1 and phage clone PCNA/λ as a template, obtaining amplified DNAfragments. Direct sequencing of the obtained amplification fragments wascarried out to determine the nucleotide sequences of the gene. Withregard to the 5′ end and 3′ end of the gene, primer λ L1 (SEQ ID NO:41)was synthesized from the nucleotide sequence in the left arm of the λvector, and λ R1 (SEQ ID NO:42) was synthesized from the nucleotidesequence in the right arm of the λ vector. These primers were used withthe primers PCNA-f2 (SEQ ID NO:43) and PCNA-r2 (SEQ ID NO:44), whichwere newly synthesized inside the gene. Phage direct PCRs were performedusing a primer pair of λL1 or λR1 and PCNA-f2 or PCNA r2 respectively,and phage clone PCNA/λ as a template, obtaining amplified DNA fragmentsincluding the 5′ end and 3′ end respectively. Similarly, directsequencing of the amplified fragments including the 5′ end and 3′ endrespectively was carried out to determine the nucleotide sequences ofthe 5′ end region and the 3′ end region of the gene.

Similar operations to those described above were performed using each ofthe genes RFCS and RFCL. In detail, either the primer λ L1 (SEQ IDNO:41) or λ R1 (SEQ ID NO:42), and one of the newly synthesized primersRFCS-f2 (SEQ ID NO:45), RFCS-r2 (SEQ ID NO:46), RFCL-f2 (SEQ ID NO:47)and RFCL-r2 (SEQ ID NO:48) were synthesized and phage direct PCR wasperformed. Thus, DNA fragments including the 5′ end region and DNAfragments including the 3′ end region were obtained, and entirenucleotide sequences were determined by direct sequencing.

The KOD-PCNA gene (SEQ ID NO:3) consists of 750 bases, and codes for 249amino acids (SEQ ID NO:2). The KOD-RFCL gene (SEQ ID NO: 5) consists of1500 bases, and codes for 499 amino acids (SEQ ID NO: 4). The KOD-RFCSgene (SEQ ID NO: 7) consists of 2601 bases, and codes for 866 aminoacids (SEQ ID NO: 6).

The homology comparison results of nucleotide sequence and protein aminoacid sequence between KOD-PCNA and PCNA derived from Archaea are shownin Table 1 below. The homology comparison results of nucleotide sequenceand protein amino acid sequence between KOD-RFCS and RFCS derived fromArchaea are shown in Table 2 below. The homology comparison results ofnucleotide sequence and protein amino acid sequence between KOD-RFCL andRFCL derived from Archaea are shown in Table 3 below.

TABLE 1 Comparison with PCNA derived from Archaea PCNA derived fromThermococcus kodakaraensis KOD1 ORF: 750 bp, 249 aa Estimated molecularweight of monomer: 28.2 kD Origin DNA length A.A. length Pfu 71.1% 750 b84.3% 249 aa Pho 69.7% 750 b 83.5% 249 aa Tfu 83.1% 750 b 91.2% 249 aaPfu: P. furiosus, Pho: P. horikoshii, Tfu: T. fumicolans

TABLE 2 Comparison with RFCS gene derived from Archaea RFCS derived fromThermococcus kodakaraensis KOD1 ORF: 981 bp, 326 aa Estimated molecularweight: 37.2 kD Origin DNA length A.A. length Afu 61.9% 960 b 58.7% 319aa Mth 63.9% 966 b 60.7% 321 aa Afu: A. fulgidus, Mth: M.thermoautotrophicum

TABLE 3 Comparison with RFCL derived from Archaea RFCL derived fromThermococcus kodakaraensis KOD1 ORF: 1500 bp, 499 aa Estimated molecularweight: 57.2 kD Origin DNA length A.A. length Pfu 67.5% 1440 b 71.5% 479aa Pho 67.8% 1412 b 74.6% 469 aa Pfu: P. furiosus, Pho: P. horikoshii

Example 3-3 Construction of Mature KOD-RFCS Gene

KOD-RFCS takes a in which the conserved region III of RFCS is divided,and an intervening sequence (KOD-RFCS Intein), which consists of 1620bases (539 amino acids), is inserted into this portion. The structuresof the RFCS and RFCL genes are shown in FIG. 20, and a comparison of theamino acid sequence of RFCS is shown in FIG. 21. This interveningsequence was compared with that derived from Archaea which also have anintervening sequence in RFCS and found a high degree of homology, 60-75%homology at the DNA level and 58-71% homology at the amino acid level.The results are shown in Table 4 below.

TABLE 4 Homology comparison of RFCS Intein RFCS derived fromThermococcus kodakaraensis KOD1 ORF: 1620 bp, 539 aa Estimated molecularweight: 52.5 kD Origin DNA Identities A.A. Identities Pho 75% 626/84071% 383/539 Pab 65% 331/509 56% 302/539 Mja 60% 510/857 58% 322/548 Pho:P. horikoshii probable replication factor C subunit Pba: P. abbysireplication factor C, small chain Mja: M. jannaschii replication factorC homolog

This sequence was deleted by the In-Fusion PCR cloning method.

Two fragments from which the intervening sequence had been deleted wereamplified by the In-Fusion PCR cloning method using a phage clone as atemplate and each of 2 pairs of primers: NdeI-mRFCS and ΔRFCS-r; andΔRFCS-f and mRFCS-XbaI (SEQ ID Nos:49 and 50, and 51 and 52,respectively). The primers used for PCR were designed so that thesequence identical to the fragment to be bound was located on the endbound to the fragment. The primers which include the positionscorresponding to the 5′ end and 3′ end of the RFCS gene were designed tohave additional sites of NdeI recognition site and XbaI recognitionsite, respectively. The two amplified DNA fragments were mixed andsubjected to PCR again, obtaining the mature KOD-RFCS (KOD-mRFCS) genefrom which the intervening sequence had been deleted and which had anXbaI recognition site at the 5′ end and an NdeI recognition site at the3′ end. The construction procedure of the mature RFCS expression vectoris shown in FIG. 22.

Example 3-4 Construction of Recombinant Expression Vectors for PCNA,RFCS and RFCL

Primers NdeI-PCNA and PCNA-XbaI (SEQ ID Nos:53 and 54) were designed tohave additional sites of an NdeI recognition site and an XbaIrecognition site at the 5′ end and 3′ end, respectively, of KOD-PCNAgene whose nucleotide sequence had been determined. Similarly, primersNdeI-RFCL and RFCL-XbaI (SEQ ID Nos:55 and 56) were designed to haveadditional sites of an NdeI recognition site and an XbaI recognitionsite at the 5′ end and 3′ end, respectively, of the KOD-RFCL gene. PCRwas performed by using these primer pairs and a phage clone includingthe full length of each of these as a template, obtaining a KOD-PCNAgene and KOD-RFCL gene, both of which have an NdeI recognition site atthe 5′ end and an XbaI recognition site at the 3′ end.

NdeI/NheI recognition sites of pET and the restriction enzymerecognition sites of these DNA fragments were used for subcloning,obtaining recombinant expression vectors (pET-PCNA, pET-mRFCS,pET-RFCL). The XbaI and NheI cut ends have compatible sites, enablingtheir ligation

Subsequently, the nucleotide sequences of the inserted DNA fragments inplasmids pET-PCNA, pET-mRFCS and pET-RFCL were determined by the dideoxychain termination method to confirm that there was no mutation resultingfrom PCR. The structure of the PCNA expression vector is shown in FIG.23, and the structure of the mRFCS and RFCL expression vectors are shownin FIG. 24.

Example 3-5 Construction of mRFCS-RFCL Recombinant Coexpression Vector

PCR was performed by using pET-RFCL as a template and phosphorylatedprimers pET-f (SEQ ID NO:57) and RFCL-SpeI (SEQ ID NO:58). The obtainedDNA fragments were cut with restriction enzyme SpeI, obtaining RFCL genefragments having a T7 promoter/ribosome binding site upstream of thegene. PCR was performed by using pET-mRFCS as a template, NdeI-mRFCS(SEQ ID NO:49) and a phosphorylated primer mRFCS-XbaI (SEQ ID NO:52).The obtained DNA fragments were cut with the restriction enzyme NdeI,obtaining RFCS gene fragments. The NdeI/NheI recognition site of pET andthe two gene fragments obtained was simultaneously ligated, obtaining acoexpression vector (pET-mRFCS-RFCL). SpeI and NheI cut ends havecompatible sites, enabling their ligation.

Subsequently, the nucleotide sequences of the inserted DNA fragments inplasmid pET-mRFCS-RFCL were determined by the dideoxy chain terminationmethod to confirm that there was no mutation resulting from PCR. Thestructure of the mRFCS-RFCL coexpression vector is shown in FIG. 25.

Example 3-6 Expression and Purification of KOD-PCNA

E. coli BL21 (DE3) was transformed with the recombinant expressionvector pET-PCNA obtained in Example 3-4. The obtained transformant wascultured in LB medium (Molecular Cloning, p.A. 2,1989). The induction ofthe T7 promoter was carried out by addingisopropyl-β-D-thiogalactopyranoside 2 hours before the harvest. Cellswere harvested from the culture medium by centrifugation. The cells wereresuspended in a buffer solution, and then pressure-disrupted, obtaininga cell extract. The extract of cell disruption was heated at 80° C. for30 minutes to insolubilize unpurified proteins derived from the hostcell. After the insoluble fraction was removed by centrifugation, thesupernatant was applied to two columns of HiTrapQ Cr. and Superdex 200Cr., obtaining a purified PCAN sample derived from KOD1. The PCNApurification procedure is shown in FIG. 26(A), and the SDS-PAGE patternsof KOD and PCNA are shown in FIG. 26(B).

After the obtained purified sample was subjected to SDS-PAGE andtransferred to a PVDF film, desired sites were cut out from the film andthe N end sequences of the samples were determined by a proteinsequencer. The result of sequencing was P-F-E-V-V, showing consistencywith M-P-F-E-V-V which was presumed from the nucleotide sequence of thegene. Results are shown in Table 5 below.

TABLE 5 Determination of N end sequence of KOD accessory proteinPresumption from DNA Peptide sequence PCNA M-P-F-E-V-V P-F-E-V-V RFCLM-T-E-V-P-W M-T-E-V-P RFCS M-S-E-E-V-K S-E-E-V-K

Example 3-7 Expression of KOD-mRFCS and KOD-RFCL

E. coli BL21 (DE3) was transformed with the recombinant expressionvectors pET-mRFCS and pET-RFCL obtained in Example 3-4. The obtainedtransformant was cultured in LB medium. The induction of the T7 promoterwas carried out by adding isopropyl-β-D-thiogalactopyranoside 2 hoursbefore the harvest. Cells were harvested from the culture medium bycentrifugation. The cells were resuspended in a buffer solution, andthen ultrasonically disrupted, obtaining a cell extract. The extract ofcell disruption was heated at 80° C. for 30 minutes to insolubilizeunpurified proteins derived from the host cell. The insoluble fractionwas removed by centrifugation, obtaining crudely purified samples ofRFCL and RFCS.

Example 3-8 Expression and Purification of KOD-RFC Complex (mRFCS-RFCL)

E. coli BL21 (DE3) was transformed with using the recombinant expressionvector pET-mRFCS-RFCL obtained in Example 3-5. The obtained transformantwas cultured in LB medium. The induction of the T7 promoter was carriedout by adding isopropyl-β-D-thiogalactopyranoside 2 hours before theharvest. Cells were harvested from the culture medium by centrifugation.The cells were resuspended in a buffer solution, and thenpressure-disrupted, obtaining a cell extract. The extract of celldisruption was heated at 80° C. for 30 minutes to insolubilizeunpurified proteins derived from the host cell. The solution was thencentrifuged and the supernatant of disrupted cell was collected. Inaddition, nucleic acids were removed with polyethyleneimine and theinsoluble fraction was removed by centrifugation. The supernatant wasapplied to two columns (Hydroxyapatite Cr., HiTrapQ Cr.), obtaining apurified sample of RFC complex (mRFCS-RFCL) derived from KOD1. Thepurification procedure of the RFC complex is shown in FIG. 27(A), andthe SDS-PAGE pattern of the RFC complex is shown in FIG. 27(B).

The obtained purified samples were subjected to SDS-PAGE and transferredto a PVDF film, and the desired sites of mRFCS and RFCL were cut outfrom the film. The N terminal sequences of the samples were determinedby a protein sequencer. Sequencing results were as follows: mRFCS:S-E-E-V-K, and RFCL: M-T-E-V-P. These showed consistency with thesequences presumed from the nucleotide sequence of the gene, i.e.,mRFCS: M-S-E-E-V-K, and RFCL: M-T-E-V-P-W. The results are shown inTable 5 above.

Example 3-9 Association Ratios of KOD-PCNA Complex and the KOD-RFCComplex

The association ratio of KOD-PCNA complex was presumed from itsmolecular weight per monomer presumed from KOD-PCNA gene and from thepreviously reported association ratio of Pfu-PCNA derived fromhyperthermophilic bacteria Pyrococcus furiosus, the presumed value being84.6 kD

The association ratio of KOD-RFC complex was presumed from its molecularweight per monomer presumed from KOD-mRFCS and KOD-RFCL, and from thepreviously reported association ratio of Pfu-RFC complex derived fromhyperthermophilic bacteria Pyrococcus furiosus, the presumed valuesbeing 206 kD.

A purified PCNA sample and a purified RFC sample were mixed, and themixture was analyzed with TSKgel G3000SW (TOSOH CORPORATION), giving achart shown in FIG. 28. Their molecular weights determined by comparisonwith standard samples were as follows: PCNA: 87.1 kD, RFC complex: 227.4kD. These values are both similar to the values estimated from thegenes. It is thus deduced that PCNA has a homotrimer structure; the RFCcomplex has a heteropentamer structure; and mRFCS and RFCL areassociated at a ratio of 1:4 in the RFC complex. The results are shownTable 6 below.

TABLE 6 Association conditions deduced from the molecular weights ofPCNA and RFC Monomer molecular Number of weight associations PresumedMeasured deduced in value value from gene P. furiosus (kDa) (kDa) PCNA28.2 3 84.6 87.1 RFC L 57.2 1 206 227.4 S 37.2 4

Example 3-10 Effect of KOD-PCNA Protein for DNA Polymerase

150 fmol of KOD DNA polymerase was reacted with 1.5 pmol of DNA obtainedby annealing an M13 P7 primer (SEQ ID NO:59) to M13 mp18 DNA (circularsingle-stranded DNA) to determine processivity. 450 fmol, 1.5 pmol, 4.5pmol and 15 pmol samples of KOD-PCNA obtained in Example 3-6 were addedto reaction buffer solutions [20 mM Tris-HCl (pH 7.5 at 75° C.), 10 mMKCl, 6 mM (NH₄)₂SO₄, 2 mM MgCl₂, 0.1% Triton X-100, 10 μg/ml BSA]. Themixtures were reacted at 75° C. for varying times: 30 seconds, 60seconds and 120 seconds, to investigate their degrees of elongation. Inthe course of the elongation reaction DNA samples were withdrawn at theabove reaction times and added to the same quantity of a stop solution(60 mM EDTA, 60 μM NaOH, 0.1% BPB, 30% glycerol). The obtained DNAsamples were separated by 1% alkaline agarose electrophoresis andanalyzed to determine the sizes of the synthesized DNA. Theelectrophoresis patterns of the synthesized DNA are shown in FIG. 29.

The patterns of the samples with no PCNA added showed 1.2 kb elongationwith a reaction time of 30 seconds, 2.5 kb elongation with a reactiontime of 60 seconds, and 5 kb elongation with a reaction time of 120seconds. The pattern showed no point at which the DNA polymerase and thesubstrate specifically dissociated. The processivity of KOD DNApolymerase is supposed to be 5 kb or more. In the state of the substratebeing in excess, i.e., 1.5 pmol of the substrate DNA relative to 150fmol of DNA polymerase, the elongation rate by KOD DNA polymerase wasabout 40 bases per second.

It was found that the addition of PCNA tended to increase the DNAelongation rate. The DNA elongation rate was highest, i.e., 120 basesper second, when PCNA was added in 30 times the molar amount of DNApolymerase. This value is about 3 times as high as that of the samplewith no PCNA added.

It is thought that a signal was observed in the vicinity of 0.5-2 kbbecause part of the KOD DNA polymerase molecule was bound to PCNA andthus were unable to contribute to promoting DNA synthesis activation. Itis thought that an excess PCNA, as much as several ten times the amountof KOD DNA polymerase, was necessary because PCNA is in annular form andthus is difficult to act on annular single stranded DNA. Accordingly,RFC, which is thought to participate in opening and closing of PCNA, wasfurther added to the PCNA to conduct a similar investigation and confirmwhether this low molecular weight signal would disappear.

Example 3-11 Effect for DNA Polymerase of the Copresence of KOD-PCNAProtein and KOD-RFC Complex

KOD DNA polymerase reactions were conducted in a similar manner toExample 3-10 either in the presence of KOD-PCNA or in the copresence ofKOD-PCNA and KOD-RFC by using an circular single stranded DNA, which wasannealed with a primer, as a substrate. The results are shown in FIG.30. As with the results of Example 3-10, when only PCNA was added, anincrease in the DNA elongation rate was observed, and a signal wasobserved in the vicinity of 0.5-2 kb. In contrast, when RFC and PCNAtogether were added, the signal at about 0.5-2 kb was diminished. Thisis presumably because almost all the KOD DNA polymerase molecules wereavailable to react with PCNA in the presence of the RFC complex. Thissuggests that the three kinds of proteins are working cooperatively. Theelectrophoresis patterns of the synthesized DNA are shown in FIG. 30.

As seen from FIG. 30, compared with the reaction using only KOD DNApolymerase, the DNA elongation rate was increased in the copresence ofPCNA and RFC. The amount of synthesized DNA was more than twice as muchin the copresence of both PCNA and RFC.

Example 3-12 Effect of KOD-PCNA Protein on PCR

The effect produced by the addition of KOD-PCNA protein on PCRamplification of a 3.6 kb DNA in a human μ-globin cluster wasinvestigated. The PCR buffer supplied with KOD-Plus (TOYOBO CO.,LTD),0.2 mM dNTPs and 0.3 μM of a pair of primers of SEQ ID Nos:60 and 61were used. 1 U KOD DNA polymerase was used as a heat-resistant DNApolymerase, and genomic DNA derived from human cell line K562 was usedas a template. This reaction mixture was subjected to PCR usingGeneAmp2400 (PE Applied Biosystems) to investigate the difference in thePCR results caused by the presence or absence of PCNA. The PCRconditions were as follows: 1 preliminary reaction of 94° C. for 2minutes; and then 35 cycles of 94° C. for 20 seconds, 60° C. for 30seconds and 68° C. for 4 minutes. The electrophoresis patterns of thePCR products are shown in FIG. 31.

When no PCNA was added and 3 ng of the template DNA was used, slightamplification of the target DNA was observed within a smear. Incontrast, amplification of the target DNA was more clearly observed byadding PCNA, despite the amount of the template being the same. When 30fmol of PCNA was added to the reaction solution, amplification of thetarget DNA was observed with the addition of 1 ng of the template DNA.That is, PCR sensitivity was increased at least 3-fold by the additionof PCNA. The amount of PCNA at which the effect of the addition occursis not limited to this concentration since it will vary depending on thekind of the enzyme used, target DNA, PCR buffer, etc.

Example 3-13 Effect of KOD-RFC Complex on PCR

The effect produced by the addition of the KOD-RFC complex wasinvestigated with the same target DNA and reaction solution as inExample 3-12. The amount of RFC effective for PCR was investigate byusing 1 ng of genomic DNA derived from human cell line K562 as atemplate.

The electrophoresis patterns of the PCR products are shown in FIG. 32.As seen from FIG. 32, amplification of the target DNA was not observedwhen no RFC was added, but it was observed when 750 fmol of RFC wasadded to the reaction solution. The amount of RFC at which the effect ofthe addition occurs is not limited to this concentration since it willvary depending on the kind of the enzyme used, target DNA, PCR buffer,etc.

Example 3-14 Effect of the Copresence of KOD-PCNA Protein and KOD-RFCComplex for PCR

The effect produced by the addition of KOD-PCNA protein and KOD-RFCcomplex was investigated with the same target DNA and reaction solutionas in Example 3-12. 1 ng of genomic DNA derived from human cell lineK562 was used as a template and their optimal ratios were investigated.The electrophoresis patterns of the PCR products are shown in FIG. 33.As seen from FIG. 33, when 10 fmol of PCNA alone was added to thereaction solution, amplification of the target DNA was barely observedtogether with a smear, but sufficient amplification was observed when1.5 pmol of PFC was further added to this reaction solution.

The amount ratio of KOD-PCNA protein to the KOD-RFC complex at which theeffect of the addition occurs is not limited to these concentrationratios since it will vary depending on the kind of the enzyme used,target DNA, PCR buffer, etc.

Example 3-15 Effect for the Copresence of KOD-PCNA Protein and theKOD-RFC Complex in PCR Using a Mixed-Type DNA Polymerase

The effect produced by the combined use of KOD-PCNA protein and theKOD-RFC complex in PCR using a mixture of two kinds of DNA polymeraseswas investigated with the same target DNA and reaction solution as inExample 3-12

1 ng of genomic DNA derived from human cell line K562 was used as atemplate. KOD Dash DNA polymerase, which is a mixture of KOD DNApolymerase and KOD (exo-) DNA polymerase which has lost KOD 3′-5′exonuclease activity, was used as the mixed-type DNA polymerase.

The electrophoresis patterns of the PCR products are shown in FIG. 34.As seen from FIG. 34, when no PCNA or RFC was added, amplification ofthe target DNA was barely observed together with a smear. In contrast,amplification of the target DNA was made somewhat clearer by theaddition of PCNA, and amplification of the target DNA was greatlyclarified by the further addition of RFC. The amount ratio of KOD-PCNAprotein to the KOD-RFC complex at which the effect of the additionoccurs is not limited to these concentration ratios since it will varydepending on the kind of the enzyme used, target DNA, PCR buffer, etc.

INDUSTRIAL AVAILABILITY

By using the composition for enhancing synthesis of DNA of theinvention, hitherto impossible target nucleic acid DNA syntheses becomepossible. In addition, not only simple DNA synthesis but also PCRsuccess rate can be enhanced. By using the DNA polymerase-related factorof the invention, the DNA synthesis activity of a DNA polymerase can beenhanced. Therefore, these are suitable for use in DNA synthesismethods, DNA amplification methods, nucleotide sequencing methods, etcsuch as PCR.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows the electrophoresis pattern of the PCR products obtained inExample 1-1.

FIG. 2 shows the electrophoresis pattern of the PCR products obtained inExample 1-2.

FIG. 3 shows the electrophoresis pattern of the PCR products obtained inExample 1-3.

FIG. 4 shows the electrophoresis pattern of the PCR products obtainedusing a primer pair of SEQ ID NOs: 10 and 11 in Example 1-4.

FIG. 5 shows the electrophoresis pattern of the PCR products obtainedusing a primer pair of SEQ ID NOs: 12 and 13 in Example 1-4.

FIG. 6 shows the electrophoresis pattern of the PCR products obtained inExample 1-5.

FIG. 7 shows the electrophoresis pattern of the PCR products obtained inExample 1-6.

FIG. 8 shows the electrophoresis pattern of the PCR products obtained inExample 1-7.

FIG. 9 shows the electrophoresis pattern of the PCR products obtained inExample 1-8.

FIG. 10 shows the electrophoresis pattern of the PCR products obtainedin Example 1-9.

FIG. 11 shows the electrophoresis pattern of the PCR products obtainedin Example 2-1.

FIG. 12 shows the electrophoresis pattern of the PCR products obtainedin Example 2-2.

FIG. 13 shows the electrophoresis pattern of the PCR products obtainedin Example 2-3.

FIG. 14 shows the electrophoresis pattern of the PCR products obtainedin Example 2-4.

FIG. 15 shows the electrophoresis pattern of the PCR products obtainedin Example 2-5.

FIG. 16 shows the electrophoresis pattern of the PCR products obtainedin Example 2-6.

FIG. 17 shows the electrophoresis pattern of the PCR products obtainedin Example 2-7.

FIG. 18 shows the electrophoresis pattern of the PCR products obtainedin Example 2-8.

FIG. 19 shows the procedure for cloning the gene encoding theKOD1-derived DNA polymerase-related factor in Example 3-1.

FIG. 20 shows the structure of the RFCS and RFCL genes used in Example3-3.

FIG. 21 shows a comparison between the RFCS amino acid sequences of Mthand KOD in Example 3-3.

FIG. 22 shows the procedure for constructing the mature RFCS expressionvector produced in Example 3-3.

FIG. 23 shows the PCNA expression vector produced in Example 3-4.

FIG. 24 shows the mRFCS and RFCL expression vectors produced in Example3-4.

FIG. 25 shows the mRFCS-RFCL coexpression vector produced in Example3-5.

FIG. 26(A) shows the PCNA purification procedure performed in Example3-6. FIG. 26(B) shows the electrophoresis pattern showing the purity ofthe PCNA thus obtained.

FIG. 27(A) shows the RFC purification procedure performed in Example3-8. FIG. 27(B) shows the electrophoresis pattern showing the purity ofthe RFC thus obtained.

FIG. 28 shows the HPLC patterns of PCNA and RFC in Example 3-9.

FIG. 29 shows the electrophoresis pattern of the DNAs obtained inExample 3-10.

FIG. 30 shows the electrophoresis pattern of the DNAs obtained inExample 3-11.

FIG. 31 shows the electrophoresis pattern of the PCR product obtained inExample 3-12.

FIG. 32 shows the electrophoresis pattern of the PCR product obtained inExample 3-13.

FIG. 33 shows the electrophoresis pattern of the PCR product obtained inExample 3-14.

FIG. 34 shows the electrophoresis pattern of the PCR product obtained inExample 3-15.

1. A method for enhancing synthesis of DNA comprising (a) providing amixture comprising a template nucleic acid and components for enzymaticDNA synthesis of the template nucleic acid, wherein the componentscomprise an enzyme with DNA polymerase activity, (b) adding to themixture at least one carboxylate ion-supplying substance that iseffective in promoting DNA synthesis in enzymatic DNA synthesisreactions, wherein the carboxylate ion-supplying substance is selectedfrom the group consisting of oxalic acid, malonic acid, esters of oxalicacid, esters of malonic acid, salts of malonic acid, and esters ofmaleic acid, and (c) maintaining the mixture to provide DNA synthesis ofthe template nucleic acid.
 2. The method of claim 1, wherein thecarboxylate ion-supplying substance is oxalic acid or malonic acid. 3.The method of claim 1, wherein the carboxylate ion-supplying substanceis a malonic acid salt.
 4. The method of claim 1, wherein thecarboxylate ion-supplying substance is an oxalic acid ester, a malonicacid ester or a maleic acid ester.
 5. The method of claim 1, wherein themethod further comprises adding to the mixture at least one compoundselected from the group consisting of dimethylsulfoxide and compoundsrepresented by the following formulaR²—CH₂—NR¹ _(x)H_(y)  (1) wherein R¹ is an alkyl group having 1 to 3carbon atoms, R² is a substituent selected from the group consisting ofthe following (a) and (b): (a) ═O (oxygen) and (b) radicals representedby the formula

wherein R⁴ is methyl or hydrogen and forms a pyrrolidine ring whencombined with R¹, R⁵ is —CO₂H or —SO₃H, and n is an integer from 0 to 2,x is an integer from 1 to 3 and y is an integer from 0 to 2, providedthat x plus y equals
 3. 6. The method of claim 5, wherein the compoundof formula (1) is trimethylglycine.
 7. The method of claim 5, whereinthe mixture contains the compound of formula (1) in an amount of 0.5 to2M and/or dimethylsulfoxide in an amount of 0.1 to 15 wt. %.
 8. Acomposition for synthesizing DNA comprising at least one carboxylateion-supplying substance that is effective in promoting DNA synthesis inenzymatic DNA synthesis reactions and an enzyme having DNA polymeraseactivity, wherein the carboxylate ion-supplying substance is selectedfrom the group consisting of oxalic acid, malonic acid, esters of oxalicacid, esters of malonic acid, salts of malonic acid, and esters ofmaleic acid.
 9. The composition of claim 8, wherein the enzyme havingDNA polymerase activity is a DNA-directed DNA polymerase.
 10. Thecomposition of claim 9, wherein the DNA-directed DNA polymerase isselected from the group consisting of Taq polymerase, Tth polymerase,Tli polymerase, Pfu polymerase, Pfutubo polymerase, Pyrobest polymerase,Pwo polymerase, KOD polymerase, Bst polymerase, Sac polymerase, Ssopolymerase, Poc polymerase, Pab polymerase, Mth polymerase, Phopolymerase, ES4 polymerase, VENT polymerase, DEEPVENT polymerase, EX-Taqpolymerase, LA-Taq polymerase, Expand polymerases, Platinum Taqpolymerases, Hi-Fi polymerase, Tbr polymerase, Tfl polymerase, Trupolymerase, Tac polymerase, Tne polyrnerase, Tma polymerase, Tihpolymerase, Tfi polymerase, and variants, modified products andderivatives thereof.
 11. The composition of claim 9, wherein theDNA-directed DNA polymerase is a thermostable DNA-directed DNApolymerase which synthesizes DNA at a rate of at least 30 bases/secondand has 3′-5′ exonuclease activity.
 12. The composition of claim 9,wherein the DNA-directed DNA polymerase is a thermostable DNA-directedDNA polymerase which synthesizes DNA at a rate of at least 30bases/second and which exhibits an error rate of 4% or less whenperforming PCR using pMol 21 as a template.
 13. The composition of claim8, wherein the enzyme having DNA polymerase activity is a reversetranscriptase.
 14. The composition of claim 13, wherein the reversetranscriptase is an enzyme selected from the group consisting of AMV-RTpolymerase, M-MLV-RT polymerase, HIV-RT polymerase, EIAV-RT polymerase,RAV2-RT polymerase, C. hydrogenoformans DNA polymerase, SuperScript I,SuperScript II, and variants, modified products and derivatives thereof.15. The composition of claim 13, wherein the reverse transcriptase is anenzyme with substantially reduced RnaseH activity.
 16. The compositionof claim 8, wherein the composition further comprises at least onemember selected from the group consisting of nucleotides, nucleotidederivatives, buffers, salts, template nucleic acids and primers.
 17. Thecomposition of claim 16, wherein the nucleotides aredeoxyphosphonucleotides and nucleotide derivatives aredeoxyphosphonucleotide derivatives.
 18. The composition of claim 17,wherein the deoxyphosphonucleotides and derivatives thereof are selectedfrom the group consisting of dATP, dCTP, dGTP, dTTP, dITP, dUTP,α-thio-dNTPs, biotin-dUTP, fluorescein-dUTP and digoxigenin-dUTP.
 19. Amethod for synthesizing DNA comprising the steps of: (a) mixing atemplate nucleic acid with the composition of claim 8, a nucleotideand/or a nucleotide derivative, and primers to form a mixture; and (b)incubating the mixture under such conditions that DNA is synthesized ata rate of at least 30 bases/second and, when performing PCR using pMol21 as a template, the error rate is 4% or less to prepare a firstnucleic acid molecule complementary to the entire or part of thetemplate nucleic acid.
 20. The method of claim 19 further comprising thestep of (c) incubating a mixture containing the first nucleic acidmolecule under such conditions that DNA is synthesized at a rate of atleast 30 bases/second and, when performing PCR using pMol 21 as atemplate, the error rate is 4% or less to prepare a second nucleic acidmolecule complementary to the entire or part of the first nucleic acidmolecule.
 21. The method of claim 19 using hot start PCR.
 22. A DNAamplification method comprising the steps of: (a) mixing a templatenucleic acid with the composition of claim 8, nucleotide and/ornucleotide derivatives and primers to form a mixture; and (b) incubatingthe mixture under such conditions that DNA is synthesized at a rate ofat least 30 bases/second and, when performing PCR using pMol 21 as atemplate, the error rate is 4% or less to amplify a nucleic acidmolecule complementary to the entire or part of the template nucleicacid.
 23. The method of claim 22 further comprising the step of (c)incubating a mixture containing the first nucleic acid molecule undersuch conditions that DNA is synthesized at a rate of at least 30bases/second and, when performing PCR using pMol 21 as a template, theerror rate is 4% or less to prepare a second nucleic acid moleculecomplementary to the entire or part of the first nucleic acid.
 24. Themethod of claim 22 using hot start PCR.
 25. A method for nucleotidesequencing comprising the steps of: (a) mixing a target nucleic acidwith the composition of claim 8, nucleotide and/or nucleotidederivatives, primers and a release factor to form a mixture; (b)incubating the mixture under such conditions that DNA is synthesized ata rate of at least 30 bases/second and, when performing PCR using pMol21 as a template, the error rate is 4% or less to amplify a nucleic acidmolecule complementary to the entire or part of the target nucleic acid;and (c) separating the amplified nucleic acid molecule to determine theentire or partial nucleotide sequence.
 26. The method of claim 25further comprising, between steps (b) and (e) (or between steps (b) and(d) when the process further comprises step (d)), the step of (c)incubating a mixture containing a first nucleic acid molecule under suchconditions that DNA is synthesized at a rate of at least 30 bases/secondand, when performing PCR using pMol 21 as a template, the error rate is4% or less to prepare a second nucleic acid molecule complementary tothe entire or part of the first nucleic acid molecule.
 27. The method ofclaim 25 using hot start PCR.
 28. A kit for synthesizing DNA comprisinga DNA polymerase and a thermostable DNA polymerase-related factorderived from a hyperthermophilic archaeon, Thermococcus kodakaraensis,which promotes the DNA synthesis activity of a DNA polymerase, whereinthe thermo stable DNA polymerase-related factor is selected from thegroup consisting of: (a) a protein comprising the amino acid sequence ofSEQ ID NO: 6; and (b) a protein comprising an amino acid sequenceencoded by the nucleotide sequence of SEQ ID NO:
 7. 29. A method forsynthesizing DNA comprising (a) providing a mixture comprising atemplate nucleic acid, at least one carboxylate ion-supplying substancethat is effective in promoting DNA synthesis in enzymatic DNA synthesisreactions, and an enzyme having DNA polymerase activity, wherein thecarboxylate ion-supplying substance is selected from the groupconsisting of oxalic acid, malonic acid, esters of oxalic acid, estersof malonic acid, salts of malonic acid, and esters of maleic acid, and(b) maintaining the mixture to provide DNA synthesis of the templatenucleic acid.
 30. The method of claim 29, wherein the enzyme having DNApolymerase activity is a DNA-directed DNA polymerase.
 31. The method ofclaim 30, wherein the DNA-directed DNA polymerase is selected from thegroup consisting of Taq polymerase, Tth polymerase, Tli polymerase, Pfupolymerase, Pfutubo polymerase, Pyrobest polymerase, Pwo polymerase, KODpolymerase, Bst polymerase, Sac polymerase, Sso polymerase, Pocpolymerase, Pab polymerase, Mth polymerase, Pho polymerase, ES4polymerase, VENT polymerase, DEEPVENT polymerase, EX-Taq polymerase,LA-Taq polymerase, Expand polymerases, Platinum Taq polymerases, Hi-Fipolymerase, Tbr polymerase, Tfl polymerase, Tru polymerase, Tacpolymerase, Tne polymerase, Tma polymerase, Tih polymerase, Tfipolymerase, and variants, modified products and derivatives thereof. 32.The method of claim 30, wherein the DNA-directed DNA polymerase is athermostable DNA-directed DNA polymerase which synthesizes DNA at a rateof at least 30 bases/second and has 3′-5′ exonuclease activity.
 33. Themethod of claim 30, wherein the DNA-directed DNA polymerase is athermostable DNA-directed DNA polymerase which synthesizes DNA at a rateof at least 30 bases/second and which exhibits an error rate of 4% orless when performing PCR using mMOl 21 as a template.
 34. The method ofclaim 29, wherein the enzyme having DNA polymerase activity is a reversetranscriptase.
 35. The method of claim 34, wherein the reversetranscriptase is an enzyme selected from the group consisting of AMV-RTpolymerase, M-MLV-RT polymerase, HIV-RT polymerase, EIAV-RT polymerase,RAV2-RT polymerase, C. hydrogenoformans DNA polymerase, SuperScript I,SuperScript II, and variants, modified products and derivatives thereof.36. The method of claim 34, wherein the reverse transcriptase is anenzyme with substantially, reduced RnaseH activity.
 37. The method ofclaim 29, further comprising adding at least one member selected fromthe group consisting of nucleotides, nucleotide derivatives, buffers,salts, template nucleic acids and primers.
 38. The method of claim 37,wherein the nucleotides are deoxyphosphonucleotides and nucleotidederivatives are deoxyphosphonucleotide derivatives.
 39. The method ofclaim 38, wherein the deoxyphosphonucleotides and derivatives thereofare selected from the group consisting of dATP, dCTP, dGTP, dTTP, dITP,dUTP, α-thio-dNTPs, biotin-dUTP, fluorescein-dUTP and digoxigenin-dUTP.40. The composition of claim 8, wherein the carboxylate ion-supplyingsubstance is selected from the group consisting of malonic acid, estersof malonic acid, and salts of malonic acid.
 41. The composition of claim40, wherein the carboxylate-ion-supplying substance is selected from thegroup consisting of zinc oxalate, ammonium oxalate, potassium oxalate,calcium oxalate, diethyl oxylate, N,N′-disuccinimidyl oxalate, dimethyloxalate, tin oxalate, cerium oxalate, iron oxalate, copper oxalate,sodium oxalate, nickel oxalate, bis oxalate, 2,4-dinitrophenyl oxalate,2,4,6-trichiorophenyl oxalate, manganese oxalate, methyl oxalate,lanthanum oxalate, lithium oxalate, isoproplylidene malonate, ethylmalonate, diethyl malonate, dibenzyl malonate, dimethyl malonate,thallium malonate, and disodium malonate.
 42. The composition of claim8, wherein the composition further comprises at least one compoundselected from the group consisting of dimethylsulfoxide and compoundsrepresented by the following formulaR²—CH₂—NR¹ _(x)H_(y)  (1) wherein R¹ is an alkyl group having 1 to 3carbon atoms, R² is a substituent selected from the group consisting ofthe following (a) and (b): (a) ═O (oxygen) and (b) radicals representedby the formula

wherein R⁴ methyl or hydrogen and forms a pyrrolidine ring when combinedwith R¹, R⁵ is —CO₂H or —SO₃H, and n is an integer from 0 to 2, x is aninteger from 1 to 3 and y is an integer from 0 to 2, provided that xplus y equals 3.